Oral delivery of angiotensin converting enzyme 2 (ace2) or angiotensin-(1-7)-bioencapsulated in plant cells attenuates pulmonary hypertension, cardiac dysfunction and development of autoimmune and experimentally induced ocular disorders

ABSTRACT

Emerging evidence indicates that diminished activity of the vasoprotective axis of the renin-angiotensin system, constituting angiotensin converting enzyme2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to pulmonary hypertension (PH). However, clinical success for long-term delivery of ACE2 or Ang-(1-7) would require stability and ease of administration to increase patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect from acids and gastric enzymes; fusion to a transmucosal carrier facilitates effective systemic absorption. Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) attenuated monocrotaline (MCT)-induced increase in right ventricular systolic pressure, decreased pulmonary vessel wall thickness and improved right heart function in both prevention and reversal protocols. Furthermore, combination of ACE2 and Ang-(1-7) augmented the beneficial effects against cardio-pulmonary pathophysiology induced by MCT administration. 
     Experiments have also been performed which indicate that this approach is also suitable for the treatment or inhibition of experimental uveitis and autoimmune uveoretinitis These studies provide proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary and ocular disease therapeutics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/001,667, filed Aug. 24, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/425,243, filed May 29, 2019, which is adivisional of U.S. patent application Ser. No. 15/030,377, filed Apr.18, 2016, which is a continuation-in-part application ofPCT/US2014/061428, filed Oct. 20, 2014, which in turn claims the benefitof U.S. Provisional Application Nos. 61/892,717, 61/943,754 and61/952,078 filed Oct. 18, 2013, Feb. 24, 2014 and Mar. 12, 2014,respectively. Each of these applications is incorporated herein byreference as though set forth in full.

STATEMENT OF FEDERAL FUNDING

This invention was made with government support under grant numbersHL102033, HL 106687, EY021752 EY21721, and FR:109442 awarded by theNational institutes of Health. The U.S. Government has certain rights inthe invention.

REFERNCE TO SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingsubmitted via EFS-Web as a text file named SEQLISTrev.txt, created Feb.2, 2022 and having a size of 29,536 bytes.

BACKGROUND OF THE INVENTION

Numerous publications and patent documents, including both publishedapplications and issued patents, are cited throughout the specificationin order to describe the state of the art to which this inventionpertains. Each of these citations is incorporated herein by reference asthough set forth in full.

Pulmonary hypertension (PH) is a devastating lung disease characterizedby elevated blood pressure in the pulmonary circulation, whicheventually leads to right-heart failure and death.¹ Although significantadvances have been made in recent years to improve the quality of lifeof patients with PH; none of the current treatments are successful inreversing PH or decreasing mortality. This has led to the realizationthat novel mechanism based therapies must be developed to accomplishthis goal.²

It is well-recognized that activation of the vasodeleterious axis of therenin angiotensin system (RAS), comprising of angiotensin-convertingenzyme (ACE), angiotensin II (AngII) and angiotensin type I receptor(AT1R) is involved in the development of PH.^(3.4) However, the clinicaluse of ACE inhibitors or AT1R blockers have yielded mixed results,thereby failing to reach a consensus opinion regarding their use for PHtherapy. Nonetheless, the recent discovery of a close homolog of ACE,angiotensin converting enzyme2 (ACE2) has resulted in the re-evaluationof the role of RAS in PH.^(5,6) ACE2 is widely expressed in the lungs,⁷predominantly on the pulmonary vascular endothelium, and catalyzes theconversion of AngII to Angiotensin-(1-7) [Ang-(1-7)]. Ang-(1-7) is avasoactive heptapeptide that mediates its effects by stimulating the Masreceptor.⁸ Thus, ACE2-Ang-(1-7)-Mas receptor constitutes thevasoprotective axis of RAS, which counterbalances the deleteriousactions of the ACE-AngII-AT1R axis.

Recent reports indicate that decreased tissue and circulating levels ofACE2 are associated with lung diseases in humans.^(9,10) On the otherhand, restoration of ACE2 through genetic overexpression, administrationof recombinant protein or use of pharmacological ACE2 activatorsresulted in cardiopulmonary protective effects against animal models ofpulmonary diseases.¹¹⁻¹⁵ These findings provided compelling evidence forinitiating clinical trials with recombinant ACE2 or Ang-(1-7) intreating pulmonary disorders. Although clinical trials are currentlyunderway (ClinicalTrials.gov; NCT01884051), the cost of manufacturing,protein stability, repetitive intravenous dosing and patient compliancepose major impediments in realizing full therapeutic potential of thistherapy.

The renin-angiotensin system (RAS) plays an important role not only inthe cardiovascular homeostasis, but also in the pathogenesis ofinflammation and autoimmune dysfunction in which Angiotensin II (Ang II)functions as the potent proinflammatory effector via Angiotensin Type 1receptor (AT1 receptor). Most components of RAS have been identified inevery organ including the eye. The tissue-specific RAS is believed toexert diverse physiological effects locally independent of circulatingAng II (Paul et al., (2006) Physiol Rev. 86:747-803). Several studieshave shown that ACE2/Ang-(1-7)/Mas axis also influences inflammatoryresponses and negatively modulates leukocyte migration, cytokineexpression and release, and fibrogenic pathways (Qui et al. (2014)Invest,. OPthalmol Vis Sci. 55:3809-3818) We have recently shown thatincreased expression of ACE2 and Ang-(1-7) reduced diabetes-inducedretinopathy and inflammation in both mouse and rat models of diabeticretinopathy (Rawas-Qalaji et al., (2012) Curr Eye Res. 37:345-356),activation of endogenous ACE2 activity reduced endotoxin-induced uveitis(Kwon et al., (2013)Adv. Drug Deliv Rev 65:782-799), providing theproof-of-concept that enhancing the protective axis of RAS is apromising therapeutic strategy for ocular inflammatory diseases.

However, the ability to deliver drugs efficiently to the retina or thebrain remains a key challenge due to anatomic barriers and physiologicalclearance mechanisms [13].

SUMMARY OF INVENTION

In accordance with the present invention a composition comprisinglyophilized plant material comprising a therapeutic protein produced inchloroplasts which retains biological function in lyophilized form isprovided. Surprisingly oral administration of said material to a patientin need thereof is effective to produce a beneficial therapeutic result.In one embodiment of the invention, the plant material comprises leavesobtained from a homoplasmic plant, and the therapeutic protein is afusion protein comprising angiotensin-converting enzyme 2 (ACE-2), andcholera non toxic B subunit (CTB) and exerts beneficial cardioprotectiveeffects. In another embodiment, the plant material comprises leavesobtained from a homoplasmic plant, and the therapeutic protein is afusion protein comprising angiotensin-(1-7) (Ang-(1-7)), and cholera nontoxic B subunit (CTB), and provides a cardioprotective effect. The plantspecies for transgenic expression of said therapeutic protein caninclude, without limitation, lettuce, carrots, cauliflower, cabbage,grass, low-nicotine tobacco, spinach, kale, and cilantro. The fusionproteins described above can contain a hinge peptide and furin cleavagesite between said CTB and said ACE-2 or said Ang1(1-7).

In a particularly preferred embodiment, the lyophilized plant materialcomprises a combination of ACE-2 and angiotensin-(1-7). In anotheraspect of this embodiment, the ACE2 sequence is codon optimized and isencoded by SEQ ID NO: 24.

In another aspect, a method for the treatment of pulmonary hypertensioncomprising administration of an effective amount of the compositionsdescribed above to a patient in need thereof is the disclosed whereinthe composition exerts a cardioprotective effect comprising at least oneof improved right heart function, decreased pulmonary vessel wallthickness, and a lack of altered basal system blood pressure, saidmethod optionally comprising assessing said effects after administrationof said composition. In certain embodiments, the patient is assessed forcardioprotective effects.

In another aspect of the invention, the compositions described above canbe used to advantage to reduce ocular inflammation. Thus, the presentinvention also provides a method for treating or delaying the onset ofuveoretinitis in a subject in need thereof comprising oraladministration of a therapeutically effective amount of the compositionsdescribed above, wherein the administration is effect to reduce ocularinflammation in said subject, the method optionally comprising assessingsaid reduction in ocular inflammation in said subject. In certainembodiments, the subject is assessed for a reduction in ocularinflammation.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A -1G. Characterization, concentration and evaluation ofpentameric structure of CTB-ACE2 and CTB-Ang-(1-7) expressed in plantchloroplasts. (FIG. 1A) Schematic representation of CTB-ACE2 andCTB-Ang-(1-7) gene cassettes and flanking regions. Southern blotanalysis of (FIG. 1B) CTB-ACE2 and (FIG. 1C) CTB-Ang-(1-7)transplastomic lines. HindIII-digested untransformed (UT) andtransformed (lane 1, 2, and 3) genomic DNA was probed with P³²-labeledflanking sequence. Quantification of (FIG. 1D) CTB-ACE2 and (FIG. 1E)CTB-Ang-(1-7) as a percentage of the total leaf proteins (TLP). (FIG.1F) GM1 binding assay of CTB-ACE2 and CTB-Ang-(1-7). CTB, non-toxiccholera B subunit (ing); UT, untransformed wild type; F, fresh; L,lyophilized; BSA, bovine serum albumin (1%, w/v). (FIG. 1G) Western blotanalysis of CTB-Ang-(1-7) in non-reducing condition without boiling andDTT. Lanes 1, 2, and 3: 10, 15, and 20 ng of CTB; total homogenate ofCTB-Ang-(1-7): 0.2, 0.4, 0.8, and 1.6 μg. The pentameric structures forthe CTB alone and the fusion protein are indicated by arrow head andarrow, respectively. Data shown are mean±SD of three independentexperiments.

FIGS. 2A-2H Oral administration of bioencapsulated ACE2 or Ang-(1-7)prevents MCT-induced PH. (FIG. 2A) Measurement of right ventricularsystolic pressure (RVSP) in normal controls and MCT-challenged rats thatwere either untreated or orally fed with wild type leaf material orgavaged with bioencapsulated ACE2/Ang-(1-7).(FIG. 2B) Right ventricle(RV) hypertrophy, measured as the ratio of RV to left ventricle (LV)plus interventricular septum (S) weights [RV/(LV+S)]. Measurement of(FIG. 2C) right ventricular end-diastolic pressure (RVEDP), (FIG.2D)+dP/dt, and (FIG. 2E)−dP/dt. Echocardiography data representing (FIG.2F) ejection fraction (EF), (FIG. 2G) ratio of the right to left enddiastolic area, signifying right heart dilation and (FIG. 2H) the bloodflow rate in the right ventricular outflow tract (RVOT). Data shown aremean±SEM. ***P<0.001 vs. control rats and #P<0.05 vs. untreated or wildtype leaf fed MCT-rats. n=6-8 animals/group.

FIGS. 3A-3D. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) improvescardiac function in PH as measured by echocardiography. Echocardiographywas performed at the end of the study in M mode at the parasternal shortaxis view at the papillary muscle level as described in the Methods.Ejection fraction of the left ventricle was calculated using thefollowing formula: (End Diastolic Volume−End Systolic Volume/EndDiastolic Volume)×100. In the parasternal short axis view, thetransducer was slightly angled to record the image of both right andleft ventricle and this image was used to analyze the right and leftventricular end diastolic area. (FIG. 3A) End Diastolic Area of LeftVentricle (LV EDA), as visualized by echocardiography. LV EDA wassignificantly reduced in PH animals and the oral feeding of ACE2 orAng-(1-7) prevented the reduction in the LV EDA. (FIG. 3B) End DiastolicArea of Right Ventricle (RV EDA), as visualized by echocardiography. RVEDA was significantly increased in the PH animals demonstrating theright ventricular dilation. Oral feeding of ACE2 or Ang-(1-7)significantly reduced the RV dilation and improved the cardiac function.(FIG. 3C) Ejection Fraction of Left Ventricle, as visualized byechocardiography. LV EF was slightly reduced in PH animals. Datarepresents mean±SEM with n=5 animals. (FIG. 2D) Ejection fraction ofRight Ventricle, as visualized by echocardiography. RV EF wassignificantly reduced in the PH animals demonstrating the cardiacdysfunction. Oral feeding of ACE2 or Ang-(1-7) attenuated the MCTinduced decrease in RV ejection function. (MCT+ACE2-P andMCT+Ang-(1-7)-P represents the prevention protocol, while MCT+ACE2-R andMCT+Ang-(1-7)-R represents the reversal protocol. MCT+C-500 andMCT+C-250 represent the combination treatment using 500mg and 250mgrespectively in the reversal protocol.) In, both figures, data representmean±SEM with n=5 animals *** denoting p<0.001 as compared with normalcontrols, while # representing p<0.05 Vs untreated and wild type plantmaterial fed MCT animals as assessed by One-Way ANOVA followed byNewman-Keuls test.

FIG. 4. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) increasescirculating levels of Ang-(1-7) in both prevention and reversalprotocols. (A) Data indicates significant elevation in circulatinglevels of Ang-(1-7) in both prevention and reversal protocols (n=5 ratsper group). (MCT+ACE2 (P) and MCT+Ang-(1-7) (P) represents theprevention protocol, while MCT+ACE2 (R) and MCT+Ang-(1-7) (R) representsthe reversal protocol. Data represent mean ±SEM with n=5 animals **denoting p<0.01 as compared with normal controls, untreated and wildtype plant material fed MCT animals as assessed by One-Way ANOVAfollowed by Newman-Keuls test.

FIGS. 5A -5D. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) exertscardioprotective effects. (FIG. 5A) Data indicates significant elevationof right ventricular systolic pressure (RVSP) after 2-weeks of MCTadministration, signifying induction of PH (n=5 rats per group). Dataare expressed as mean±SEM; * p<0.05 versus controls using studentt-test. Measurement of (FIG. 5B) right ventricular end-diastolicpressure (RVEDP), (FIG. 5C) +dP/dt, and (FIG. 5D) -dP/dt in in normalcontrols and MCT-challenged rats that were either untreated or orallyfed with wild type leaf material or gavaged with bioencapsulatedACE2/Ang-(1-7). Data are expressed as mean±SEM; ***p<0.001; versuscontrols and #p<0.05 versus MCT group. n=5 animals per experimentalgroup.

FIGS. 6A-6C. Oral feeding of bioencapsulated ACE2 or Ang-(1-7) exertsanti-fibrotic and anti-remodeling effects in the prevention protocol.(FIG. 6A) Interstitial collagen deposition in the right ventricle. (FIG.6B) Staining for α-smooth muscle actin to quantify medial wall thicknessof the pulmonary arteries measuring less than 50 μm. Scale bar denotes10 μm (FIG. 6C) ACE2 activity was measured in rat sera (10 μl) collectedfrom different experimental groups AFU: Arbitrary fluorescence units.Data represents mean±SEM with * denoting p<0.05 Vs other groups, **denoting p<0.01 as compared with controls, while # representing p<0.05vs. untreated and wild type plant material fed MCT-rats as assessed byOne-Way ANOVA followed by Newman-Keuls test.

FIGS. 7A -7G. Oral treatment with ACE2 or Ang-(1-7) arrests diseaseprogression and attenuates cardiopulmonary remodeling. (FIG. 7A)Individual values of the right ventricle systolic pressure (RVSP) fromdifferent experimental groups of the reversal protocol. (FIG. 7B)RV/(LV+S) values from individual animals, denoting right hearthypertrophy. Echocardiography data representing (FIG. 7C) ratio of theright to left ventricle end diastolic area, (FIG. 7D) ejection fraction(EF), and (FIG. 7E) the blood flow rate in the right ventricular outflowtract (RVOT) of the different experimental groups. (FIG. 7F)Representative photographs and quantification of interstitial fibrosis(FIG. 7G) Measurement of vessel wall thickness following a-smooth muscleactin staining of the pulmonary arteries (<50 μm). Scale bar denotes 10μm. Data shown are mean±SEM. ** P<0.01, ***P<0.001 vs. control rats and#P<0.05 vs. untreated or wild type leaf fed MCT-rats. n=6-8animals/group.

FIG. 8A-8H. Combination therapy with ACE2 and Ang-(1-7) rescuesestablished PH. (FIG. 8A) Measurement of right ventricular systolicpressure (RVSP) in MCT rats treated with a combination of either 500 mgor 250 mg each of ACE2 and Ang-(1-7). (FIG. 8B) Data representing rightventricular hypertrophy as a ratio of RV/(LV+S). Measurement of (FIG.8C) right ventricular end-diastolic pressure (RVEDP), (FIG. 8D)+dP/dt,and (FIG. 8E)−dP/dt from the combination study. Echocardiography datarepresenting (FIG. 8F) ejection fraction (EF), (FIG. 8G) ratio of theright to left end diastolic area and (FIG. 8H) the blood flow rate inthe right ventricular outflow tract (RVOT). Data shown are mean±SEM.***P<0.001 vs. control rats and #P<0.05 vs. untreated or wild type leaffed MCT-rats. n=6-8 animals/group.

FIGS. 9A -9B. Combination of ACE2 and Ang-(1-7) decreases ventricularfibrosis and attenuates pulmonary vascular remodeling. (FIG. 9A)Representative photographs of collagen staining and quantitativeanalysis of right ventricular fibrosis following 2-week treatment withcombination therapy. (FIG. 9B) Measurement of vessel wall thickness ofthe pulmonary arteries (<50 μm). Scale bar denotes 10 μm. Data areexpressed as mean±SEM; **p<0.01; versus controls and # p<0.05 versusuntreated and wild type plant material fed MCT-rats. n=5-7 animals perexperimental group.

FIGS. 10A -10J. Effects of ACE2 or Ang-(1-7) treatment on the lungrenin-angiotensin system (RAS), pro-inflammatory cytokines and autophagy(prevention protocol). Relative change in lung mRNA levels of (FIG. 10A)Angiotensin-converting enzyme (ACE), (FIG. 10B) angiotensin-convertingenzyme 2 (ACE2), (FIG. 10C) ACE/ACE2 ratio, (FIG. 10D) AT1R, (FIG. 10E)AT2R and (FIG. 10F) AT1R/AT2R receptor. Relative mRNA levels of lungpro-inflammatory cytokines, (FIG. 10G) tumor necrosis factor (TNF)-α,(FIG. 10H) transforming growth factor (TGF)-β and (FIG. 10I) toll-likereceptor-4 (TLR-4) from the MCT study. Autophagy marker, LC3-II isincreased in the lungs of MCT-exposed animals. (FIG. 10J) Immunoblot anddensitometry analysis of the lung LC3I/II protein expression. Data areexpressed as mean±SEM. * P<0.05, ** P<0.01, and *** P<0.001 versuscontrol rats. #P<0.05 versus MCT group.

FIGS. 11A-11J. Effects of Monotherapy as well as the combination therapyon the lung RAS components, pro-inflammatory cytokines and autophagy inthe reversal protocol. Data represent relative changes in lung mRNAlevels of (FIG. 11A) Angiotensin-converting enzyme (ACE), (FIG. 11B)angiotensin-converting enzyme 2 (ACE2), (FIG. 11C) ACE/ACE2 ratio, (FIG.11D) Angiotensin type 1 receptor (AT1R), (FIG. 11E) Angiotensin type 2receptor (AT2R) and (FIG. 11F) AT1R/AT2R ratio. Relative mRNA levels oflung pro-inflammatory cytokines, (FIG. 11G) tumor necrosis factor(TNF)-α, (FIG. 11H) transforming growth factor (TGF)-β and (FIG. 111)toll-like receptor-4 (TLR-4) from the same study. (FIG. 11J) Immunoblotand densitometry quantification showing lung LC3I/II protein expression.Data are expressed as mean±SEM. * p<0.05 and ** p<0.01 versus controlrats, while #P<0.05 versus MCT group.

FIGS. 12A -12E. Evaluation of proper formation of pentameric structureand lyophilization for the CTB fusion proteins. FIG. 12A. Western blotanalysis to investigate the proper folding and assembly of CTB-Ang-(1-7)expressed in chloroplasts. Four micrograms of total leaf protein wereloaded for each lane with (+) or without (−) treatment of denaturingagents. CTB, purified non-toxic cholera B subunit (10 ng); UT,untransformed wild type; DTT, 100 mM; boiling, incubation of samples inboiling water for 3 min; H, homogenate total leaf protein; S,supernatant fraction after centrifugation of the total leaf protein;Arrows and numbers, locations of monomer and oligomers of CTB-Ang-(1-7);P, pentamer-pentamer complexes. FIG. 12B. Western blot analysis for thecomparison of the level of CTB-Ang-(1-7) in lyophilized (L) and fresh(F) leaves. Equal amount of lyophilized and fresh leaf material (10 mg)was extracted in same volume (300 μl) of extraction buffer. 1Xrepresents 1 μl of homogenate protein resuspended in extraction buffer.The samples were boiled in DTT prior to loading on SDS acrylamide gel.Purified CTB standard protein was loaded as indicated for densitometricanalysis. FIG. 12C. and FIG. 12D. Comparison of the level ofCTB-Ang-(1-7) and -ACE2 in lyophilized (L) and fresh (F) leaves. Dataare means±SD of three independent experiments. FIG. 12E. GM1 bindingassay of CTB-ACE2 and -Ang-(1-7). Extracted total protein samples wereserially diluted up to 10 pg/ul, which means 11 on the Y axis, and usedfor GM1 binding assay. The binding affinity was read at 450 nm then anabsorbance of >0.1 after background signal substraction was determinedas positive. Two and three different batches were examined for fresh andlyophilized leaf materials, respectively, and indicated as blackdiamond. CTB, purified non-toxic cholera B subunit (black circle); UT,untransformed wild type (black triangle); F, fresh; L, lyophilized; 12and 15, 12- and 15-month storage at room temperature. *p<0.001 (versusfresh); #p<0.001 (versus WT).

FIGS. 13A -13D. ACE2 activity assay using protein samples extracted fromCTB-ACE2 transplastomic and untransformed leaf materials (WT). FIG. 13A.Assay buffer containing the substrate was also used as a negativecontrol. FIG. 13B. Increased ACE2 in both serum and retina in mice fedwith CTB-ACE2 leaf material detected by Western blotting using ananti-ACE2 polyclonal antibody. FIG. 13C. ACE2 activities in serum andretina from mice fed with either fresh (F, 500 mg/ mouse), orlyophilized (L, 50 mg/mouse) CTB-ACE2 leaf materials, compared to micefed with wild type (WT) leaf materials; (n=5 per group). The experimentwas repeated at least twice with similar results. * p<0.05 (versus WTleaf). FIG. 13D. A graph showing results from an ELISA assaydemonstrating the presence of Ang-(1-7) in plasma and retina after oraladministration.

FIGS. 14A -14C. Histological evaluation of EIU mice. The mice wereorally administered with Wild-type, CTB-ACE2 and CTB-Ang-(1-7) expressedleaf material for five days before LPS (25 ng/eye) injection. Eyes wereenucleated 24hr after LPS injection, fixed and processed for paraffinsections and stained with H&E. FIG. 14A. Representative photographs ofthe iris ciliary body, anterior chamber and posterior chamber. Originalmagnifications 20×. Bar=50 μm. FIG. 14B. Histopathologic scoreevaluation. Inflammatory cells per section in the iris ciliary body,anterior chamber and posterior chamber were counted from H&E stainedparaffin sections from eyes at 24 h after EIU induction. Values ony-axis represent no. of infiltrating inflammatory cells/section. Resultsare given as mean+SD; (n=6 per group); *P<0.05 (versus WT+LPS group).FIG. 14C. Histological evaluation of EIU mice. The mice were orallyadministered with different doses of lyophilized plant cells expressingCTB-ACE2 for four days before LPS (25 ng/eye) injection. Eyes wereenucleated 24 hr after LPS injection, fixed and processed for sectionsand stained with H&E and Histopathologic score was evaluated by at leasttwo individuals. Inflammatory cells per section in the iris ciliarybody, anterior chamber and posterior chamber were counted from H&Estained sections from eyes at 24 h after EIU induction. Values on y-axisrepresent no. of infiltrating inflammatory cells/section. Results aregiven as mean+SD; (n=6 per group); *P<0.05 (versus WT+LPS group).

FIGS. 15A-15B. Real-time reverse transcriptase (RT)-PCR analysis ofocular mRNA levels of inflammatory cytokines (FIG. 15A) and RAS genes(FIG. 15B). Values on y-axis represent fold difference compared toage-matched wild-type control ocular samples for each gene. WT ctrl,non-fed wild-type control; WT+LPS, WT leaf fed & LPS injected; ACE2+LPS,CTB-ACE2 expressed leaf fed & LPS injected; Ang-(1-7)+LPS, CTB-Ang-(1-7)expressed leaf fed & LPS injected. Data expressed as mean +SD; (n=4 pergroup); * P<0.05 (versus WT+LPS group).

FIGS. 16A -16G. Clinical evaluation of EAU from fundoscopic photographs.EAU was induced in B10. RIII mice by immunization with IRBP in CFA. Thefundoscopic images were obtained on day 14 after immunization.Representative fundus image from WT leaf fed mice (FIG. 16a , FIG. 16b); CTB-ACE2 expressed leaf fed mice (FIG. 16c , FIG. 16d ); andCTB-Ang-(1-7) expressed leaf fed mice (FIG. 16e , FIG. 16f ). FIG. 16G.Clinical EAU scores. Clinical EAU score was evaluated on a scale of 0-4.Values on y-axis represent the average of clinical scores given onfundus images. Results are given as mean+SD; (n=5 per group); *P<0.05(versus WT fed group).

FIGS. 17A -17B. Assessment of retinal thickness on OCT images from EAUmice. Horizontal and cross sectional OCT images were obtained on day 14after immunization. The retinal thickness was measured and averaged fromfive different frames of horizontal OCT scan images of single eye. FIG.17A. Representative fundus projection (left panel) and B-scan (rightpanel) images from WT leaf fed mice; CTB-ACE2 expressed leaf fed mice;and CTB-Ang-(1-7) expressed leaf fed mice. FIG. 17B. Retinal thicknessmeasured from OCT images. Values on y-axis represent the average ofretinal thickness calculated manually from B-scan OCT images. Resultsare given as mean +SD; (n=5 per group); *P<0.05 (versus WT fed group).

FIGS. 18A-18C. Histological evaluation of EAU. H&E staining,magnifications 4×, Bar=200 μm; and 20×, Bar=50 μm. FIG. 18A.Representative micrographs from animals fed with WT leaf fed mice,CTB-ACE2, and CTB-Ang-(1-7) leaf materials. Images of histologicalanalysis show severe retinal folding, loss of the photoreceptor layerand massive inflammatory cell inflammation in the vitreous, retina andsubretinal space in WT leaf fed group; moderate to minimum infiltration,photoreceptor damage, retinal folding was observed in CTB-ACE2 expressedleaf fed group; a minor infiltration of cells and retinal folding wasobserved in the CTB-Ang-(1-7) leaf fed group. FIG. 18B. Histopathologyscores. CTB-ACE2 and CTB-Ang-(1-7) leaf fed groups showed a reduced EAUhistological grade compared to controls fed with WT leaf. FIG. 18C.Evaluation of infiltrating inflammatory cells in the posterior chamber.Inflammatory cells/section in the posterior chamber were counted on 14thday after EAU induction. Values on y-axis represent no. of infiltratinginflammatory cells/section. Results are given as mean+SD; (n=5 pergroup); *P<0.05 (versus WT leaf fed group).

FIGS. 19A-19C. Evaluation of EAU from fundoscopic photographs, OCT andhistopatholgy. EAU was induced in B 10.RIII mice by immunization withIRBP in CFA. The treatment with CTB-Ang-(1-7) was delayed. Thefundoscopic images were obtained on day 14 after immunization. FIG. 19A.Clinical EAU scores. Clinical EAU score was evaluated on a scale of 0-4.FIG. 19B. Retinal thickness measured from OCT images. Horizontal andcross sectional OCT images were obtained on day 14 after immunization.The retinal thickness was measured and averaged from five differentframes of horizontal OCT scan images of single eye. FIG. 19C.Histopathology scores. CTB-Ang-(1-7) leaf fed groups showed a reducedEAU histological grade compared to controls fed with WT leaf. Values ony-axis represent the average of clinical scores given on fundus images.Results are given as mean +SD; (n=6 per group); *P<0.05 (versus WT fedgroup).

FIGS. 20A-20E. Codon distribution of psbA (FIG. 20A and 20B),chloroplast based codon tables, native and codon-optimized (N and O)ACE2 gene. Codon-optimized ACE2 were optimized by changing the rarecodons and codons usage frequency to resemble the chloroplast psbA gene(FIGS. 20C, 20D, and 20E). Sequence alignments of native andcodon-optimized (N and O) ACE2 gene. Any different nucleotides ofcodon-optimized sequences are marked in light grey. Nat: nativesequence; CO—N: codon-optimized sequence obtained from new version ofoptimizer; CO—O: codon-optimized sequence obtained from old version ofoptimizer.

FIGS. 21A and 21B. Native and Optimized CTB-ACE2 Constructs andSequences. FIG. 21A. Native sequence (SEQ ID NO: 23). FIG. 21B. Codonoptimized sequence (SEQ ID NO: 24) .

FIGS. 22A-22D. Selection and regeneration of CTB-ACE2 transplastomiclettuce. (FIG. 22A) transplastomic lettuce shoots undergoing first roundof selection; (FIG. 22B) second rounds of selection; CTB-ACE2 lettuceacclimated in growth chamber (FIG. 22C) and grown in greenhouse (FIG.22D).

FIG. 23. Western blot analysis of ACE2-expressed transplastomic lettuceplants. 5 μg total protein extracted from wild type (WT), nativeCTB-ACE2 expressed (N) and codon-optimized CTB-ACE2 expressed (CO) weredetected by anti-CTB antibody. CTB (5 ng) was loaded as positivecontrol. The homoplasmic lettuce plants expressing codon-optimized ACE2showed 7.7-fold higher expression than the native ACE2.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments disclosed herein provide a method for treating subjectssuffering from chronic diseases such as cardiovascular disease,cardiopulmonary disease, and other lung diseases involving pulmonaryfibrosis, diabetes-related micro-and macro-vascular diseases, metabolicsyndrome, stress-related disorders and ocular disorders. For example,studies have shown that overexpression of ACE2 or Ang-(1-7) in the lungsor its activation by small molecule activators prevents pulmonaryhypertension-induced lung pathophysiology (Shenoy et al, Curr OpinPharmacol 2011, 11:150-5. Shenoy et al, Am J Respir Crit Care Med. 2013,187(6):648-57). In addition, ACE2 activators attenuate ischemia-inducedcardiac pathophysiology (Qi et al, Hypertension. 2013, 62(4):746-52) andproduce beneficial effects on dysfunctional diabetic EPCs (Jarajapu etal, Diabetes,2013, 62, 1258-69. Since this enzyme is a protein, the onlyeffective way to increase its levels in a diseased state is toadminister it intravenously or intramuscularly. Both of these methods ofdelivery are extremely inefficient, and cost prohibitive in apre-clinical trials and subsequent use in therapeutics.Chloroplast-derived ACE2 and Ang-(1-7) should reduce cost and facilitateoral delivery of these therapeutic proteins, thus making it attractivefor clinical trials for above mentioned chronic diseases.

In Example 2, we described methods for enhancing the systemic and localactivity of the protective axis of the RAS by oral delivery of ACE2 andAng-(1-7) bioencapsulated in plant cells for conferring protectionagainst endotoxin induced uveitis (EIU) and experimental autoimmuneuveoretinitis (EAU). Both ACE2 and Ang-(1-7), fused with the non-toxiccholera B subunit B (CTB) were expressed in plant chloroplasts. Theeffects of orally delivered CTB-ACE2/Ang-(1-7) on EIU and EAU models inC57B6/J and B10. RIII mice respectively were examined. Increased levelsof ACE2 and Ang-(1-7) were observed in circulation and retina after oraladministration of CTB-ACE2/Ang-(1-7) leaf materials. Oral feeding ofmice with bioencapsulated ACE2 or Ang-(1-7) significantly reduced LPSinduced infiltration of inflammatory cells and expression ofinflammatory cytokines in the eye; this treatment also dramaticallydecreased cellular infiltration, retinal vasculitis, damage and foldingin EAU eyes. Thus, enhancing the protective axis of RAS by oral deliveryof ACE2/Ang-(1-7) bioencapsulated in plant cells provide an innovative,more efficient and cost-effective therapeutic strategy for ocularinflammation such as uveitis and autoimmune uveoretinitis.

Definitions

As used herein, the terms “administering” or “administration” of anagent, drug, or peptide to a subject includes any route of introducingor delivering to a subject a compound to perform its intended function.The administering or administration can be carried out by any suitableroute, including orally, intranasally, parenterally (intravenously,intramuscularly, intraperitoneally, or subcutaneously), rectally, ortopically. Administering or administration includes self-administrationand the administration by another.

As used herein, the terms “disease,” “disorder,” or “complication”refers to any deviation from a normal state in a subject.

As used herein, by the term “effective amount” “amount effective,” orthe like, it is meant an amount effective at dosages and for periods oftime necessary to achieve the desired result.

As used herein, the term “inhibiting” or “preventing” means causing theclinical symptoms of the disease state not to worsen or develop, e.g.,inhibiting the onset of disease, in a subject that may be exposed to orpredisposed to the disease state, but does not yet experience or displaysymptoms of the disease state.

As used herein, the term “expression” in the context of a gene orpolynucleotide involves the transcription of the gene or polynucleotideinto RNA. The term can also, but not necessarily, involves thesubsequent translation of the RNA into polypeptide chains and theirassembly into proteins.

A plant remnant may include one or more molecules (such as, but notlimited to, proteins and fragments thereof, minerals, nucleotides andfragments thereof, plant structural components, etc.) derived from theplant in which the protein of interest was expressed. Accordingly, acomposition pertaining to whole plant material (e.g., whole or portionsof plant leafs, stems, fruit, etc.) or crude plant extract wouldcertainly contain a high concentration of plant remnants, as well as acomposition comprising purified protein of interest that has one or moredetectable plant remnants. In a specific embodiment, the plant remnantis rubisco.

In another embodiment, the invention pertains to an administrablecomposition for treating or preventing pulmonary hypertension orpulmonary hypertension-induced lung pathophysiology. The compositioncomprises a therapeutically-effective amount of ACE2, Ang-(1-7),CTB-ACE2 or CTB-Ang-(1-7), or a combination thereof having beenexpressed by a plant and a plant remnant. The compositions of theinvention may also be used to advantage to treat ocular inflammation.

Methods, vectors, and compositions for transforming plants and plantcells are taught for example in WO 01/72959; WO 03/057834; and WO04/005467. WO 01/64023 discusses use of marker free gene constructs.

Proteins expressed in accord with certain embodiments taught herein maybe used in vivo by administration to a subject, human or animal in avariety of ways. The pharmaceutical compositions may be administeredorally or parenterally, i.e., subcutaneously, intramuscularly orintravenously, though oral administration is preferred.

Oral compositions of the present invention can be administered via theconsumption of a foodstuff that has been manufactured with thetransgenic plant producing the plastid derived therapeutic protein. Theedible part of the plant, or portion thereof, is used as a dietarycomponent. The therapeutic compositions can be formulated in a classicalmanner using solid or liquid vehicles, diluents and additivesappropriate to the desired mode of administration. Orally, thecomposition can be administered in the form of tablets, capsules,granules, powders and the like with at least one vehicle, e.g., starch,calcium carbonate, sucrose, lactose, gelatin, etc. The preparation mayalso be emulsified. The active immunogenic ingredient is often mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredient. Suitable excipients are, e.g., water,saline, dextrose, glycerol, ethanol or the like and combination thereof.In addition, if desired, the compositions may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, or adjuvants. In a preferred embodiment the edible plant, juice,grain, leaves, tubers, stems, seeds, roots or other plant parts of thepharmaceutical producing transgenic plant is ingested by a human or ananimal thus providing a very inexpensive means of treatment of orimmunization against disease.

In a specific embodiment, plant material (e.g. lettuce material)comprising chloroplasts capable of expressing ACE2, Ang-(1-7), CTB-ACE2or CTB-Ang-(1-7), or a combination thereof, is homogenized, lyophilizedand encapsulated. In one specific embodiment, an extract of the lettucematerial is encapsulated. In an alternative embodiment, the lettucematerial is powderized before encapsulation.

In alternative embodiments, the compositions may be provided with thejuice of the transgenic plants for the convenience of administration.For said purpose, the plants to be transformed are preferably selectedfrom the edible plants consisting of tomato, carrot and apple, amongothers, which are consumed usually in the form of juice.

According to another embodiment, the subject invention pertains to atransformed chloroplast genome that has been transformed with a vectorcomprising a heterologous gene that expresses a peptide as disclosedherein.

Of particular present interest is a chloroplast genome that has beentransformed with a vector comprising a heterologous gene that expressesone or more polypeptides selected from ACE2, Ang-(1-7), CTB-ACE2 orCTB-Ang-(1-7), or a combination thereof. In a related embodiment, thesubject invention pertains to a plant comprising at least one celltransformed to express a peptide as disclosed herein.

Reference to CTB and ACE2 or Ang-(1-7) sequences herein relate to theknown full length amino acid sequences as well as at least 12, 15, 25,50, 75, 100, 125, 150, 175, 200, 225, 250 or 265 contiguous amino acidsselected from such amino acid sequences, or biologically active variantsthereof. Typically, the polypeptide sequences relate to the known humanversions of the sequences.

Variants which are biologically active, refer to those, in the case oforal tolerance, that activate T-cells and/or induce a Th2 cell response,characterized by the upregulation of immunosuppressive cytokines (suchas IL10 and IL4) and serum antibodies (such as IgG1), or, in the case ofdesiring the native function of the protein, is a variant whichmaintains the native function of the protein. Preferably, naturally ornon-naturally occurring polypeptide variants have amino acid sequenceswhich are at least about 55, 60, 65, or 70, preferably about 75, 80, 85,90, 96, 96, or 98% identical to the full-length amino acid sequence or afragment thereof. Percent identity between a putative polypeptidevariant and a full length amino acid sequence is determined using theBlast2 alignment program (Blosum62, Expect 10, standard genetic codes).

Variations in percent identity can be due, for example, to amino acidsubstitutions, insertions, or deletions. Amino acid substitutions aredefined as one for one amino acid replacements. They are conservative innature when the substituted amino acid has similar structural and/orchemical properties. Examples of conservative replacements aresubstitution of a leucine with an isoleucine or valine, an aspartatewith a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an aminoacid sequence. They typically fall in the range of about 1 to 5 aminoacids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity of polypeptide can be found using computerprograms well known in the art, such as DNASTAR software. Whether anamino acid change results in a biologically active LecA polypeptide canreadily be determined by assaying for native activity, as described forexample, in the specific Examples, below.

Reference to genetic sequences herein refers to single- ordouble-stranded nucleic acid sequences and comprises a coding sequenceor the complement of a coding sequence for polypeptide of interest.Degenerate nucleic acid sequences encoding polypeptides, as well ashomologous nucleotide sequences which are at least about 50, 55, 60, 65,60, preferably about 75, 90, 96, or 98% identical to the cDNA may beused in accordance with the teachings herein polynucleotides. Percentsequence identity between the sequences of two polynucleotides isdetermined using computer programs such as ALIGN which employ the FASTAalgorithm, using an affine gap search with a gap open penalty of −12 anda gap extension penalty of −2.Complementary DNA (cDNA) molecules,species homologs, and variants of nucleic acid sequences which encodebiologically active polypeptides also are useful polynucleotides.

Variants and homologs of the nucleic acid sequences described above alsoare useful nucleic acid sequences. Typically, homologous polynucleotidesequences can be identified by hybridization of candidatepolynucleotides to known polynucleotides under stringent conditions, asis known in the art. For example, using the following wash conditions:2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, roomtemperature twice, 30 minutes each; then 2× SSC, 0.1% SDS, 50° C. once,30 minutes; then 2X SSC, room temperature twice, 10 minutes eachhomologous sequences can be identified which contain at most about25-30% basepair mismatches. More preferably, homologous nucleic acidstrands contain 15-25% basepair mismatches, even more preferably 5-15%basepair mismatches.

Species homologs of polynucleotides referred to herein also can beidentified by making suitable probes or primers and screening cDNAexpression libraries. It is well known that the Tm of a double-strandedDNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner etal., J. Mol. Biol. 81, 123 (1973). Nucleotide sequences which hybridizeto polynucleotides of interest, or their complements following stringenthybridization and/or wash conditions also are also usefulpolynucleotides. Stringent wash conditions are well known and understoodin the art and are disclosed, for example, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2^(nd) ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination oftemperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m)of the hybrid understudy. The T_(m) a hybrid between a polynucleotide of interest or thecomplement thereof and a polynucleotide sequence which is at least about50, preferably about 75, 90, 96, or 98% identical to one of thosenucleotide sequences can be calculated, for example, using the equationof Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_(m)=81.5° C.-16.6(log₁₀ [Na¹⁰ ])+0.41(% G+C)−0.63(% formamide)−600/1),

where 1=the length of the hybrid in basepairs. Stringent wash conditionsinclude, for example, 4 X SSC at 65° C., or 50% formamide, 4×SSC at 42°C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditionsinclude, for example, 0.2×SSC at 65° C.

The Examples set forth below are provided to illustrate certainembodiments of the invention. They are not intended to limit theinvention in any way.

EXAMPLE 1 Creation of Transplastomic Plants Expressing CTB-ACE2 andCTB-Ang-(1-7)

In the present example, we describe a low cost oral delivery system foradministering

ACE2 or Ang-(1-7) and test its efficacy in an experimental model of PH.We took advantage of transplastomic technology which enableschloroplasts to generate high levels of therapeutic proteins withinplant leaves.¹⁶⁻²¹ This technology presents minimal risk of humanpathogen or endotoxin contamination, eliminates complex proteinpurification steps, and abolishes cold chain and sterile deliveryrequirements that are commonly associated with protein therapy.²²⁻²⁴Efficacy of plant-based pharmaceuticals has been validated by the factthat FDA has recently approved the use of taliglucerase alfa (Tradename: Elelyso) in the treatment of Gaucher's disease.²⁵ This studyprovides evidence for the development of an oral delivery system toadminister ACE2 and Ang-(1-7) using transplastomic technology, anddemonstrates its efficacy in an established rat model of monocrotaline(MCT)-induced PH.

The following materials and methods are provided to facilitate thepractice of Example 1.

Chloroplast Transformation Vector Construction and Regeneration ofTransplastomic Lines

The cDNA for ACE2 (accession: NM 021804) was used as the template toclone CTB-ACE2 fusion gene into the intermediate vector. To cloneCTB-Ang-(1-7) fusion gene, CTB-containing vector was used as templatewith a forward primer specific to CTB and a reverse primer specific toCTB but including nucleotide sequence corresponding to 7 amino acid ofAng-(1-7) peptide. The sequence-confirmed chimeric gene was cloned intochloroplast transformation vector, pLD.

Southern Blot Analysis

To investigate transgene integration and homoplasmy, Southern blotanalysis was performed as previously described.¹ Total genomic DNA wasdigested with HindIIII, separated on a 0.8% agarose gel at 15 Vovernight, transferred onto nylon membrane. The 0.8-kb flanking regionprobe was generated by digesting the pUC-CT vector DNA with BamHI andBglII. The probe was labeled with dCTP using Klenow fragment (PromegaM220A) and random primers (Promega C1181). After labeling the probe, theblotted membrane was hybridized with hybridization solution [0.5 Mphosphate buffer pH 7.2, 1 mM EDTA pH 8.0, 7% (w/v) SDS, 1% (w/v) bovineserum albumin] at overnight at 65° C. then washed with 2X SSC, 0.2% SDSfor 30 min once and 1×SSC, 0.1% SDS for 15 min twice each.Radioisotope-labelled blots were exposed to X-ray film at −80° C. for 8h.

Quantification of CTB-ACE2 and CTB-Ang-(1-7) Fusion Protein

Leaves ground in liquid nitrogen was resuspened in extraction buffer[100 mM NaCl, 10 mM EDTA, 200 mM Tris-Cl pH 8.0, 0.1% (v/v) TritonX-100, 400 mM sucrose, 2 mM PMSF, and proteinase inhibitor cocktail] ina ratio of 100 mg to 300 μL, then vigorously mixed using vortex (−30 s)and sonicated twice (pulse on for 5 s and pulse off for 10 s).Homogenate protein was quantified using Bradford assay. For thequantification of CTB-ACE2 protein, ELISA was performed. Ninety-six-wellplates were coated with serially diluted CTB standard(25-12.5-6.25-3.13-1.56-0.781-0.391-0.195 pg/μL; Sigma C9903) andCTB-ACE2 proteins (4,000-8,000-16,000) in bicarbonate buffer (15 mMNa₂CO₃, 35 mM NaHCO₃, 3.08 mM NaN₃, pH 9.6), then incubated overnight at4° C. After washing the plates with 1X phosphate buffered saline (FisherIC-N2810307) containing 0.05% (v/v) Tween 20 (1× PBST), the coatedplates were blocked with 1XPBST containing 3% skim milk (PTM) for oneand half hr at 37° C. and incubated with rabbit anti-CTB polyclonalantibody (1:10,000 in PTM; GenWay 18-511-245283) overnight at 4° C. Theplates were followed by incubating with goat anti-rabbit IgG-HRPsecondary antibody (1:4,000 in PTM; Southern Biotechnology 4030-05) forone and half hr at 37° C. after washing with 1X PBST thrice. Theantibody-bound plates were washed with 1X PBST thrice and 1X PBS oncebefore adding the 100 μL of tetramethyl benzidine (TMB) solutionsubstrate (American Qualex Antibodies UCFL-05801). The reaction wasstopped by adding the 50 μL of 2N H₂SO₄ and read on a plate reader at450 nm. CTB-Ang-(1-7) was quantified using western blot anddensitometric analysis. Total homogenate protein (0.5 μg) and CTBstandards (3, 6, and 9 ng) was separated on SDS-PAGE and transferred tonitrocellulose membranes. Rabbit anti-CTB polyclonal antibody (1:10,000in PTM; GenWay) and goat anti-rabbit IgG-HRP secondary antibody (1:4,000in PTM; Southern Biotechnology) was used to detect the fusion proteins.SuperSignal West Pico Chemiluminescent Substrate (Pierce 34080) was usedfor autoradiographic detection. Then the developed films were analyzedby densitometry using Image J (IJ 1.46r; NIH). The known amounts of CTBstandard were plotted and then the protein samples were interpolated onthe graph. For the separation of proteins under non-reducing conditions,proteins were extracted as described above. The extracted proteins werecombined with tricine sample buffer (Bio-Rad 161-0730), in the absenceof reducing agents and without boiling prior to running on SDS-PAGEgels.

GM1 Binding Assay

To evaluate pentameric structure, GM1 binding assay was performed.Ninety-six-well plates were coated with monosialoganglioside-GM1 (SigmaG-7641) (3.0 μg/mL in bicarbonate buffer: 15 mM Na₂CO₃, 35 mM NaHCO₃, pH9.6) overnight at 4° C. The plates were washed with PBST thrice andblocked with PTM. After washing the plates with 1X PBST, homogenateplant protein was diluted to concentration of 0.1 μg/μL with the sameplant extraction buffer and incubated in the GM1 coated plates overnightat 4° C., along with CTB (1 ng/μL, Sigma), bovine serum albumin (1% w/vBSA) and untransformed wild type plant protein (0.1 μg/μL) as controls.The plates were blocked with PTM for one and half hr at 37° C. Afterdiscarding the PTM, rabbit anti-CTB polyclonal antibody (1:10,000 inPTM; GenWay) was incubated overnight at 4° C. Following washing threetimes with 1X PBST, goat anti-rabbit IgG-HRP secondary antibody (1:4,000in PTM; Southern Biotechnology) was incubated for one and half hr at 37°C. The plates were washed with PBST thrice and with PBS once and 100 μLof tetramethyl benzidine (TMB) solution substrate (American QualexAntibodies UCFL-05801) was added to the wells and incubated under darkfor 5 min. The reaction was stopped by adding 50 μL of 2N H₂SO₄, andread the absorbance at 450 nm using plate reader (Bio-rad Model 680).

Lyophilization

Frozen leaf tissues stored at −80° C. were crumbled into small piecesand transferred to 200 ml containers and sealed with porous 3M MilliporeMedical Tape. The plant samples were freeze-dried in vacuum at −52° C.at 0.036 mBar for three days, with the aid of VirTis BenchTop 6K freezedryer system. Lyophilized leaf material was stored in sealed containerat room temperature with silica gel.

PH Study Design

We used the monorrotaiine (MU) animal model of PH to evaluate thetherapeutic efficacy of oral feeding of ACE2, Ang-(1-7) or theircombination against disease pathogenesis. Animals were randomly assignedto respective experimental groups based on their body weights at thetime of MCI administration. The study design consisted of prevention andreversal protocols. All animal procedures were approved by theInstitutional Animal Care and Use Committee at the University of Floridaand complied with National Institutes of Health guidelines.

Gavage Feeding of MCT Rats with Bioencapsulated ACE2 or Ang-(1-7)

8-week-old male Sprague Dawley rats (Charles River Laboratories) wereinjected with a single subcutaneous dose of MCT (50 mg/kg, SigmaAldrich, USA). Control animals received an equivalent amount of sterilesaline (˜500 μL). For the prevention protocol, a subset of MCT animalswas simultaneously orally gavaged with wild type leafy material,bioencapsulated ACE2 or Ang-(1-7) [500 mg, twice daily in sterilephosphate-buffered saline (PBS)] for a period of 28 days. For thereversal protocol, ACE2, Ang-(1-7) or their combination [500 mg or 250mg each of ACE2 and Ang-(1-7)] was gavage-fed after 2 weeks of MCTadministration and continued for additional 15 days.

Echocardiography Measurement

Four weeks after MCT injection, transthoracic echocardiography wasperformed using GE vivid7 ultrasound machine with a 12-MHz transducer(GE Healthcare, N.J., USA). Rats were anesthetized with the 2%isoflurane-oxygen mixture. M-mode echocardiography was measured at theparasternal short-axis view at the level of papillary muscles. Leftventricular ejection fraction (LVEF) was calculated from the M-mode.Further, at this view, right and left ventricular end diastolic area(RVEDA and LVEDA) and right ventricular ejection fraction (RVEF) werealso measured. Pulsed Doppler recordings performed at the parasternalshort-axis view at the base of the heart to measure the right ventricleoutflow tract (RVOT V_(max)). ECG was recorded simultaneously for allthe assessments. All the recordings were performed in triplicates.Ejection fraction was obtained from both right and left ventricles andwas represented as the ratio between right and left ventricle.Similarly, end diastolic area was also represented as the ratio betweenright and left ventricle. Blood flow at the right ventricular outflowtract was represented as RVOT Vmax (m/s). Three consecutive cycles fromeach recording (totally 9 cycles) were averaged to assess eachparameter. Following the echocardiographic measurements, animals weresubjected to hemodynamic measurements.

Right Ventricular Systolic Pressure (RVSP) Measurements

The RVSP was measured in anesthetized animals [subcutaneous injection ofa mixture of ketamine (30 mg/Kg) and Xylazine (6 mg/Kg)] using afluid-filled silastic catheter, which was inserted inside the rightdescending jugular vein and advanced to the right ventricle. Thecatheter was connected to a pressure transducer that was interfaced to aPowerLab (AD Instruments, USA) signal transduction unit. The waveformwas used to confirm the positioning of the catheter in the rightventricle. RVSP, +dP/dt, −dP/dt and right ventricular end diastolicpressure (RVEDP) were obtained using the Chart program supplied alongwith the PowerLab system. For both prevention and reversal protocolsRVSP was measured after 4 weeks of MCT-challenge.

Hypertrophy and Histological Analysis

Following RVSP measurements, a thoracotomy was performed, and afterexsanguination, the heart and lungs were removed en bloc. To calculateright ventricular hypertrophy (RVH), the wet weight of RV and leftventricle plus ventricular septum (LV+S) was determined. RVH wasexpressed as the ratio of RV/[LV+S] weights. The RV was furtherprocessed for histological analysis of collagen content. Briefly, RV wasfixed in 10% neutral buffered formalin, embedded in paraffin, sectionedat 5 μm and stained with picro-sirius red. Interstitial fibrosis wasdetermined at 100X magnification using the ImageJ program from NationalInstitutes of Health, as previously described.² A minimum of 5-8separate images from different (non-overlapping) regions of the rightventricle were obtained. The results for each animal were then averagedfor subsequent statistical analysis. To carry out histologicalexamination of the lung, the left lung alone was perfused with PBSfollowed by 10% neutral buffered formalin. For measuring pulmonarymedial wall thickness, 5 μm thick lung sections were cut paraffinembedded and stained for a-smooth muscle actin (1:600, clone 1A4, SigmaAldrich, USA). Vessels with an external diameter of <50 μm wereconsidered to measure the medial wall thickness. For each rat, around 10vessels were counted and the average was calculated. The percent medialwall thickness was calculated using the formula: % Medial wallthickness=[(medial thickness×2)/external diameter]×100 (n=5 rats pergroup) Media thickness was defined as the distance between the laminaelastica interna and lamina elastica externa.

Real-Time RT-PCR Analysis

Semi-quantitative real time RT-PCR was used to determine mRNA levels ofthe renin-angiotensin system components viz. ACE, ACE2, AT1R, and AT2R,and pro-inflammatory cytokines (PICs) viz. Tumor Necrosis Factor-alpha(TNF-α), Transforming Growth Factor-beta (TGF-β) and toll-likereceptor-4 (TLR-4) as described previously.² Total RNA isolation, cDNAsynthesis and RT-PCR were performed as previously described. In brief,total RNA was isolated from punched tissues using TRIzol reagent(Invitrogen, USA) according to the manufacturer's specifications. TheRNA concentration was calculated from the absorbance at 260 nm and RNAquality was assured by 260/280 ratio. Only RNA samples exhibiting anabsorbance ratio (260/280) of >1.6 were used for further experiments.The RNA samples were treated with DNase I (Ambion, USA) to remove anygenomic DNA. First strand cDNA was synthesized from 2 μg RNA withiScript cDNA synthesis kit (Bio-Rad, USA). Real-time RT-PCR wasperformed in 384-well PCR plates using iTaq SYBR Green Super mix withROX (Bio-Rad) in triplicate using the ABI Prism 7900 sequence detectionsystem (Applied Biosystems, USA). The PCR cycling conditions were asfollows: 50° C. for 2 min, 95° C. for 3 min, followed for 45 cycles (15sat 95° C., and 1 min at 60° C.). To confirm the specific PCR product, adissociation step (15 s at 95° C., 15 s at 60° C., and 15s at 95° C.)was added to check the melting temperature. Gene expression was measuredby the AACT method and was normalized to 18S mRNA levels. The data arepresented as the fold change of the gene of interest relative to that ofcontrol animals.

Measurement of Ang-(1-7)

Circulating levels of Ang-(1-7) were measured using a commerciallyavailable EIA kit from Bachem Laboratories as per manufacturer'sinstructions.

Statistics

Prism 5 (GraphPad) was used for all analyses. Values are presented asmeans±SEM. Data were analyzed using one-way ANOVA followed by theNewman-Keuls test for multiple comparisons. P values less than 0.05 wereconsidered statistically significant.

RESULTS Creation and Characterization of CTB-ACE2 and CTB-Ang-(1-7)Expressed in Plant Chloroplasts

The native human ACE2 cDNA and synthetic Ang-(1-7) DNA sequences werecloned into the chloroplast transformation vector (pLDutr) (FIG. 1A).For efficient delivery of the proteins into circulation, a carrierprotein, cholera non-toxic B subunit (CTB), was fused to the N-terminalof both therapeutic proteins (FIG. 1A), which facilitates theirtransmucosal delivery by binding to monosialotetrahexosylgangliosidereceptors (GM1) present on the intestinal epithelial cells. Hinge(Gly-Pro-Gly-Pro; SEQ ID NO: 1) and furin cleavage site(Arg-Arg-Lys-Arg; SEQ ID NO: 2) were placed between CTB and therapeuticproteins (FIG. 1A) to eliminate steric hindrance and aid systemicrelease of these therapeutic proteins after they are internalized vialigand-receptor complex formation on the surface of epithelial cells.The expression of the fusion genes was driven by light regulated strongchloroplast psbA promoter and the transcripts were stabilized by placingthe psbA UTR at the 3′ end of the fusion genes (FIG. 1A). To select thechloroplast transformed with the fusion genes,aminoglycoside-3″-adenylyl-transferase gene (aadA), driven by thechloroplast ribosomal RNA promoter (Prrn), was incorporated into theexpression cassette to confer the transformants resistance tospectinomycin (FIG. 1A). This expression cassette was flanked by DNAsequences of isoleucyl-tRNA synthetase (trnI) and alanyl-tRNA synthetase(trnA) genes, identical to the native chloroplast genome at both flanks(FIG. 1A). The flanking sequences serve to facilitate transgeneintegration into the chloroplast genome (FIG. 1A) via double homologousrecombination. Chloroplast transformation vectors expressing the ACE2and Ang-(1-7) genes were coated onto gold particles and delivered intochloroplasts using the biolistic particle delivery system.²⁶ Thebombarded plant leaves were then grown on spectinomycin containing plantregeneration media. The shoots regenerated from the media wereinvestigated for the site specific integration of the transgenes intochloroplast genome and homoplasmy of the transgenes (absence ofuntransformed genomes) using Southern blot analysis with theradioisotope-labelled probe spanning trnl and trnA flanking sequences.²⁶HindIII-digested chloroplast genomic DNA from three independenttransplastomic lines for each transplastomic line showed two hybridizingfragments at 8.59 and 3.44 kb for CTB-ACE2 due to an internal Hind IIIsite of ACE2 (FIG. 1A) and a fragment at 9.71 kb for CTB-Ang-(1-7),which confirm the absence of untransformed chloroplast genomes (FIGS. 1Band 1C). Thus, stable integration of the transgenes was confirmed andthe homoplasmic lines were used for further studies. The confirmedhomoplasmic lines were multiplied using another round of antibioticselection under aseptic conditions. Then they were cultivated in acontrolled greenhouse for increasing biomass. CTB-ACE2 expression variedbetween 1.69% and 2.14% of the total leaf proteins (TLP) (FIG. 1D),depending upon the harvest time because this transgene is regulated bylight via the chloroplast psbA promoter. Similarly, the expression levelof CTB-Ang-(1-7) varied between 6.0% and 8.7% of TLP (FIG. 1E), atdifferent durations of illumination, reaching maximum expression at theend of the day. Hence, for performing in vivo experimental studies, thetherapeutic leaf materials were harvested at 6 pm and powdered in liquidnitrogen.

Both the therapeutic proteins were fused to the transmucosal carrier,CTB. The B subunit has a single intra-subunit disulfide bond whichstabilizes the CTB monomer.²⁵ The monomers then assemble to formring-shaped pentameric structure via inter-subunit interactionsincluding hydrogen bonds, salt bridges, and hydrophobic interactions.Upon oral administration, only the pentameric form of CTB binds to thegut epithelial GM1 receptor for internalization.²⁷ Hence, weinvestigated the proper formation of pentameric structure of the CTBfused proteins and their binding affinity to GM1 receptor usingGM1-ELISA. The binding affinity between CTB pentamers and the receptorwas measured spectrophotometrically as a function of absorbance at 450nm. The therapeutic proteins from the fresh leaf materials showedcomparable absorbance to CTB (FIG. 1F), confirming that chloroplastsform disulfide-bridges, fold, and assemble these fusion proteins. Wealso lyophilized the leaves expressing ACE2 and Ang-(1-7), and evaluatedtheir affinity to the GM1 receptor (FIG. 1F). Lyophilization not onlymaintained proper folding, disulfide bond and pentamer assembly but alsofacilitated long-term storage at room temperature (FIG. 1F).Furthermore, the Western blot assay performed under non-reducingconditions without DTT and boiling showed that there was no monomericform or cleaved fragments of CTB-Ang-(1-7) (FIG. 1G). In the Westernblot image, the major bands for pentameric assembly of CTB were detectedaround ˜50 kDa (FIG. 1G, arrow head) and the expected bands forpentameric assembly of CTB-Ang-(1-7) were detected (FIG. 1G, arrow).Therefore, these results confirm that the therapeutic proteins expressedin chloroplasts exist in an intact and pentameric form.

Oral Feeding of Bioencapsulated ACE2 or Ang-(1-7) Prevents MCT-InducedPH

Oral gavage of the frozen powdered leaves (500mg in sterile phosphatebuffered saline) from untransformed wild type, CTB-ACE2 or CTB-Ang-(1-7)transplastomic plants was performed twice daily for 4-weeks inMCT-challenged rats. MCT injection caused robust elevation in rightventricular systolic pressure (RVSP; FIG. 2A) that was associated withthe development of right ventricular hypertrophy (RVH) (FIG. 2B). Incontrast, MCT animals gavaged with either ACE2 or Ang-(1-7) showedconsiderable reduction in RVSP and RVH (FIGS. 2A and 2B). Furthermore,measurement of hemodynamic parameters in MCT animals revealed increasesin right ventricular end diastolic pressure (RVEDP; 153%), +dP/dt (88%)and −dP/dt (107%). Conversely, treatment with ACE2 or Ang-(1-7) restoredall these parameters to near-control levels (FIGS. 2C, 2D, and 2E).Echocardiography of MCT rats revealed an increase in the ratio of rightventricle to left ventricle end diastolic area (RV/LV EDA), implyingdilation of the right heart (FIG. 2F, and FIGS. 3A and 3B), which wasaccompanied with a decrease in ejection fraction (EF), measured as aratio of RV/LV EF (FIG. 2G and FIGS. 3C and 3D). In addition, the pulsedDoppler blood flow measurement revealed decreased flow rate in the rightventricle outflow tract (RVOT) (FIG. 2H). Furthermore, video of theechocardiography revealed maladaptive structural remodeling in MCT rathearts as compared with controls. However, oral delivery of ACE2 orAng-(1-7) exhibited improved cardioprotective effects. Both ACE2 andAng-(1-7) were effective in decreasing RV dilation (FIG. 2F), increasingEF (FIG. 2G) and preventing MCT-induced decrease in RVOT blood flow(FIG. 2H). These beneficial effects were associated with reduced cardiacremodeling as evidenced by echocardiography videos. Concurrently, RVfibrosis and pulmonary vessel wall thickness were also decreased (FIGS.6A and 6B). Oral ACE2 feeding was associated with ˜37% increase incirculating ACE2 activity as compared with MCT alone rats (FIG. 6C) anda two-fold increase in circulating levels of Ang-(1-7; FIG. 4).Interestingly, ACE2 or Ang-(1-7) did not alter the basal systemic bloodpressure (SBP: Control, 120+5; MCT, 123+7; MCT+ACE2, 118+2;MCT+Ang-(1-7), 116+4; n=5/experimental group).

Oral ACE2/Ang-(1-7) Treatment Arrests the Progression of Established PH

We next tested whether oral feeding of ACE2 or Ang-(1-7) after theinitiation of PH could arrest the disease-progression. We observed thattwo-weeks of MCT challenge induces significant elevation in RVSP(>45mmHg) as compared with controls (FIG. 5A) Hence, for this study,oral therapy was initiated after two-weeks of MCT challenge, and thetreatment continued for additional 15-days. This regime of treatmentwith ACE2 or Ang-(1-7) inhibited further elevation in MCT-induced RVSPand RVH (FIGS. 7A and 7B), and was associated with increased circulatinglevels of Ang-(1-7; FIG. 4). Improvements in hemodynamic parameters withregard to lowering RVEDP, decreasing+dP/dt, and reducing -dP/dt werealso observed (FIGS. 5B and 5D). In addition, ACE2/Ang-(1-7) therapydecreased RV dilation (FIG. 7C, and FIGS. 3A and 3B) and increased RVEF(FIG. 7D and FIGS. 3C and 3D), which was supported by echocardiographyvideo. Subsequently, blood flow in the RVOT was also improved (FIG. 7E).Finally, RV fibrosis and pulmonary vessel wall thickening weresignificantly attenuated in ACE2/Ang-(1-7) treated animals (FIGS. 7F and7G).

Combination Therapy with Oral ACE2 and Ang-(1-7) Feeding RescuesEstablished PH

Next, we evaluated the effects of a combination therapy with ACE2 andAng-(1-7), wherein 500 mg or 250 mg each of ACE2 and Ang-(1-7) plantmaterial was combined. Reversal protocol was followed for this study,wherein the combination therapy was initiated after two-weeks of MCTchallenge, and the treatment continued for the next 15 days. Asexpected, we observed better protective effects with the 500 mgcombination. This combination showed 18% more reduction in RVSP and 25%additional decrease in RV/(LV+S) ratio when compared with the 250 mgcombination (FIGS. 8A and 8B). Similarly, enhanced beneficial effects ofthe 500 mg combination were observed for other hemodynamic parameterssuch as RVEDP, +dP/dt, and −dP/dt (FIGS. 8C to 8E). Both doses of thecombination therapy were effective in decreasing RV dilatation (FIG. 8F,and FIGS. 3A and 3B), and increasing EF (FIG. 5G, and FIGS. 3C and 3D),which was accompanied by greater RVOT blood flow (FIG. 8H). All theseobservations were supported by echocardiography video. Consistent withthis were the improvement in RV fibrosis and pulmonary vessel wallthickness following combination therapy (FIGS. 9A and 9B).

Beneficial Effects of ACE2/Ang-(1-7) Oral Therapy Involve Inhibition ofPro-Inflammatory Cytokines and Autophagy

We demonstrate herein that oral delivery of ACE2 or Ang-(1-7) correctsRAS imbalance and inhibit pro-inflammatory cytokines. Data in FIG. 10support this hypothesis. MCT rats revealed increased pulmonary mRNAlevels of ACE and AT1R (FIG. 10A and 10D), which resulted in 8-fold and4-fold increases in the ACE/ACE2 and AT1R/AT2R ratios respectively(FIGS. 10C and 10F). Conversely, mRNA levels of ACE2 and AT2R wereincreased, while that of AT1R was decreased in the ACE2 or Ang-(1-7) fedMCT rats, resulting in decreased ACE/ACE2 and AT1R/AT2R ratios.Furthermore, MCT-challenged animals showed increased mRNA levels ofTNF-α (4-fold), TGF-β (4-fold) and TLR-4 (5-fold), all of which weremarkedly reduced by ACE2 or Ang-(1-7) treatment (FIGS. 10G, 10H and10I). Recent reports indicate that the autophagic protein degradationpathway is activated in MCT-challenged animals.' Accordingly, weobserved that the lung LC3B-II protein, an autophagy marker, wassignificantly increased in MCT-challenged rats (FIG. 10J). However, ACE2or Ang-(1-7) decreased LC3B-II levels, implying inhibition of autophagy.Similar results with respect to RAS modulation, anti-inflammatoryproperties and inhibition of autophagy were observed in the reversalprotocol with monotherapy [either ACE2 or Ang-(1-7)] or combinationtherapy (FIG. 11A to 11J).

DISCUSSION

Human ACE2 and Ang-(1-7) has been expressed within plant chloroplastsusing transplastomic technology. Oral administration of transplastomicplant material to rats attenuates PH. While previous geneticinterventions with ACE2/Ang-(1-7) have demonstrated beneficial effectsin animals,^(11,12) there are several challenges that limit the clinicaldevelopment of such approaches. The incidence of PH is increasing amongthe elderly global population, necessitating affordable medication forthe masses. While drugs made in plant cells have been approved by theFDA and are currently marketed,' targeted gene therapy is still in theexperimental stage and far away from clinical applications. Even if genetherapy is approved as a valid approach, it would still be available to<1% of the global population due to the limited expertise available inhospitals for gene therapy. In contrast, oral delivery of capsulescontaining therapeutic proteins produced in plant chloroplasts isfeasible and very much affordable. Accordingly, drug delivery is asimportant as drug discovery and this study focuses on the development ofa novel low cost delivery system for administering therapeutic proteinslike ACE2/Ang-(1-7), which have been found to be effective againstexperimental models of lung diseases, but not yet clinically approved.Injectable delivery of ACE2/Ang-(1-7) poses some unique challenges withrespect to cost of manufacturing, protein stability, cold storage, shelflife, sterile delivery and requirement of health professionals/hospitalsfor their administration. Most of these concerns are easily eliminatedby orally delivering therapeutic proteins bioencapsulated in plantcells. Currently produced injectable protein drugs are not affordable tomore than half of the global population, despite decades of optimizationof their process development. By developing an oral delivery system foradministering ACE2 and Ang-(1-7), as reported here, we have made atremendous advancement to move the field for the treatment of pulmonarydiseases forward.

ACE2 and Ang-(1-7) were expressed in plant chloroplasts as fusionproteins with CTB. Though Ang-(1-7) is not a gene product, a syntheticgene encoding for Ang-(1-7), was used in this study.²⁹ We have used CTBas the transmucosal carrier to facilitate the uptake of ACE2 andAng-(1-7) into circulation. Both CTB fusion proteins are disulfidebonded, form pentamers and properly folded, as observed for other CTBfusion proteins.^(17,18) CTB is an approved adjuvant³⁹ that has beenused in several clinical settings. Administration of CTB fused antigen(BD peptide) in humans with autoimmune eye disorders inducedimmunological tolerance by suppressing abnormal T cell reactivityagainst the peptide.⁴⁰ Also, immune suppression to autoantigens(pro-insulin a nd factor IX) linked to CTB have been observed in animalstudies following oral administration.^(19,20) Likewise, other studieshave shown immune-suppressive effects when CTB was fused to autoimmuneor allergic causative agents.⁴¹ The GM1 receptors present on intestinalepithelial cells make CTB the most appropriate carrier for transportingtherapeutic proteins into systemic circulation as this receptor iswidely distributed over the intestinal mucosa^(42,43) with a rapidturnover rate.⁴⁴

The half-life of native Ang-(1-7) is very short.^(45,46) However, inthis study, the stability of Ang-(1-7) was found to increase in sera. Inplant cells, CTB stabilizes Ang-(1-7) by formation of pentamers (FIG.1G), and thus confers protection from plant proteases. However, onlymonomers are observed in sera after delivery into the sera. While furincleavage site (NH2-R-R-K-R-COOH) should facilitate removal of CTB,efficiency of cleavage depends upon the flanking amino acid sequence ofthe fused protein.' Ang-(1-7) fused to CTB didn't provide optimal furincleavage site because it is not flanked by furin preferred basic aminoacids at N-terminal side and serine-valine at C-terminal side.Therefore, it is anticipated that furin cleavage will not be rapid orefficient. This offers greater N-terminal protection to Ang-(1-7) andextends its stability for several hours in the sera as compared withinjectable Ang-(1-7). Actually, chronic treatment with bioencapsulatedAng-(1-7) showed significant increases of the circulating levels of thepeptide (FIG. 4), which suggests that an oral gavage twice daily ofbioencapsulated Ang-(1-7) results in sustained elevated plasma levels ofAng-(1-7) in the treated animals. Ang-(1-7) concentration in frozen leafmaterials was found to be 584 μg/g, which translates to 292 μg in 500mg. This dose compares well with previous studies, wherein, 750-1000μg/Kg of Ang-(1-7) peptide was administered to rats (˜30 ug per 300 gmsrat).

Oral feeding of bioencapsulated ACE2 or Ang-(1-7) prevents thedevelopment, and most importantly, retards the progression ofMCT-induced PH. An upregulation of the deleterious ACE-AngII-AT1R axisand downregulation of the protective ACE2-Ang-(1-7)-Mas axis contributesto PH pathogenesis.⁴⁸ Thus, maintaining equilibrium between these twoaxes is crucial for preserving pulmonary vascular homeostasis. Oraldelivery of ACE2 or Ang-(1-7) increased ACE2/ACE and decreased AT1R/AT2Rratios, signifying improvement in pulmonary RAS balance. In addition,the ACE2-fed MCT rats prevented the decrease in serum ACE2 activityobserved in PH animals. Also, about 2-3 fold increase in the circulatinglevels of Ang-(1-7) in ACE2 treated animals was observed, which couldpossibly contribute to the protective effects (FIG. 4). ACE2 fed ratsexhibited 37% increase in the enzymatic activity as compared with MCTalone animals. This increase in disease treated animals restored theenzymatic activity of circulating ACE2 to that of normal healthy rats.Most importantly, this increase was sufficient to exert beneficialeffects against PH pathophysiology. Previous experimental studies haveshown that exogenous administration of recombinant ACE2 increases serumACE2 activity to exert therapeutic efficacy in several diseasemodels.⁴⁹⁻⁵¹ Increasing serum ACE2 levels is also clinically significantsince abnormally low levels of serum ACE2 have been associated withPH.¹⁰ Surprisingly, pulmonary ACE2 mRNA levels were increased with oralACE2 feeding. We speculate this increase to be a positive feed forwardmechanism, since similar increases in ACE2 have been reportedpreviously.^(14,15) Also, AT2R levels were increased with ACE2treatment, which is consistent with previous studies showing aprotective role of this receptor in cardiopulmonary disease.⁵²Furthermore, the favorable RAS modulation by ACE2 or Ang-(1-7) wasassociated with reduced lung inflammatory cytokines. Pro-inflammatorycytokines contribute to thickening of the pulmonary arterioles leadingto heightened pulmonary pressure.⁵³ In line with these findings, weobserved marked increases in vessel wall thickness in MCT-challengedanimals. However, ACE2 or Ang-(1-7) treatment significantly inhibitedmedial wall thickness. The observed effects of ACE2/Ang-(1-7) could beattributed to reduction in pro-inflammatory cytokines as well as directanti-proliferative actions on the vascular smooth muscle cells, acontention supported by earlier studies.⁵⁴ Recent studies haveimplicated autophagy in PH.²⁶ We observed an increase in LC3B-II, anautophagy marker, in MCT rats, which was significantly decreased withACE2 or Ang-(1-7) treatment.

Collectively, the aforementioned findings indicate that oral delivery ofACE2 or Ang-(1-7) corrects a dysregulated pulmonary RAS, reducesinflammation, decreases vascular remodeling and inhibits autophagy toexert lung-protective effects. Importantly, ACE2/Ang-(1-7) treatment didnot lower basal systemic blood pressure, which is important sinceinduction of systemic hypotension can be detrimental in patients withPH. Similar phenomenon has also been observed in other studies, wherein,chronic administration of Ang-(1-7) fails to decrease systemic BP in avariety of models of hypertension⁵⁵⁻⁵⁷. One possibility may be relatedto pulmonary vasculature being more sensitive to Ang-(1-7) or thatabundant receptors for Ang-(1-7) are present on the pulmonary vessels.Furthermore, a combination of ACE2 and Ang-(1-7) treatment producedbeneficial effects on the cardiopulmonary system. We observed that thehigher dose combination yielded better effects than the lower dose.

Of particular interest are the cardioprotective effects of oralACE2/Ang-(1-7) therapy. Sustained pressure overload on the right heartinduces ventricular remodeling and dysfunction.⁵⁸ Echocardiography ofMCT rats revealed prominent structural changes in the heart. The RVassumed a round shape, with a shift in the intraventricular septumcausing RV dilation with reduced EF. In addition, the pulmonary arteryflow was significantly lowered in the MCT group. All these changes wereassociated with development of RVH, increased interstitial fibrosis andcardiac dysfunction. However, ACE2 or Ang-(1-7) treatment restorednormal heart structure, inhibited RV dilatation and improved EF. Also,RVH and interstitial fibrosis were significantly reduced, along withpreserved cardiac function. Moreover, the combination therapy with ACE2and Ang-(1-7) was found to exert superior cardioprotective effects.

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EXAMPLE 2 Oral Delivery of ACE2/Ang-(1-7) Bioencapsulated in Plant CellsProtects against Experimental Uveitis and Autoimmune Uveoretinitis

The following materials and methods are provided to facilitate thepractice of Example II.

Chloroplast Transformation Vector Construction and Regeneration ofTransplastomic Lines

Performed as described above in Example I.

Animals and Experimental Procedures

Wild-type C57B1/6J mice (6-8 weeks old)) and B10.RIII mice (8-10 weeksold) were purchased from Jackson Laboratories (Bar Harbor, Me.) andmaintained at the Animal Care

Service at the University of Florida. All procedures adhered to the ARVOstatement for the use of Animals in Ophthalmic and Vision Research, andthe protocol was approved by the Animal Care and Use Committee of theUniversity of Florida. The animals were fed standard laboratory chow andallowed free access to water in an air-conditioned room with a 12-12-hrlight dark cycle.

The mice were divided in three groups for EIU model and orally gavagedwith control (untransformed wild-type, WT) tobacco leaves, CTB-ACE2, andCTB-Ang-(1-7) expressing transplastomic tobacco leaves. The mice weregiven ˜500 mg of the specified tobacco leaf material suspended insterile PBS, by careful gavage into the hypopharynx twice in a day for 5days. For preparation of the gavage material, leaves were frozen andground in liquid nitrogen. EIU was induced by a single intravitrealinjection of Escherichia coli LPS (25 ng/eye) (Sigma-Aldrich, Inc., St.Louis, Mo.) dissolved in sterile pyrogen-free saline, on the fifth dayof feeding. All animals were anesthetized and pupils were dilated beforeintraocular injections. Each experimental group included at least 4-6animals and each experiment was performed at least twice.

For the EAU model, the mice were divided in three groups and orallygavaged with ˜500 mg of the control wild-type tobacco leaves, CTB-ACE2,CTB-Ang-(1-7) expressing transplastomic tobacco leaves once daily for 15days. EAU was induced by active immunization with ˜50 μg of IRBP(161-180) (SGIPYIISYLHPGNTILHVD) (Genscript, Piscataway, N.J.) with CFA(Sigma-Aldrich, Inc., St. Louis, Mo.) (1:1 vol/vol) subcutaneously, onthe second day of feeding. Each experimental group included at least 4-6animals and each experiment was performed at least twice to ensurereproducibility.

Different Doses of Lyophilized CTB-ACE2 Plant Cell Oral Gavage andInduction of EIU

In another approach, the mice were divided in three groups and orallygavaged with varying dosage of lyophilized CTB-ACE2 expressingtransplastomic tobacco leaves. The mice were given 12.5 mg, 25 mg and 50mg of the specified lyophilized plant cells suspended in sterile PBS, bycareful gavage into the hypopharynx once in a day for 4 days EIU wasinduced by a single intravitreal injection of Escherichia coli LPS (25ng/eye) (Sigma-Aldrich, Inc., St. Louis, Mo.) dissolved in sterilepyrogen-free saline, on the fourth day of feeding. All animals wereanesthetized and pupils were dilated before intraocular injections. Eachexperimental group included at least 4-6 animals and each experiment wasperformed at least twice.

Delaying of Oral Gavage and Induction of EAU

The mice were divided in three groups and orally gavaged with ˜50 mg ofthe lyophilized plant cells expressing CTB-Ang-(1-7) once daily. Thetreatment with CTB-Ang-(1-7) started at day 5 and day 10 after IRBPinjection to induce EAU, and continued daily till dayl4. EAU was inducedby active immunization with ˜50 μg of IRBP (161-180)(SGIPYIISYLHPGNTILHVD; SEQ ID NO: 22) (Genscript, Piscataway, NJ) withCFA (Sigma-Aldrich, Inc., St. Louis, Mo.) (1:1 vol/vol) subcutaneously.Each experimental group included at least 4-6 animals and eachexperiment was performed at least twice to ensure reproducibility.

Histopathological Evaluation

The EIU mice were euthanized 24hr after LPS injection and the eyes wereenucleated immediately and fixed in 4% paraformaldehyde freshly made inPBS overnight at 4° C. and processed for paraffin embedding andsections. Sagittal sections (4 μm) from every 50 μm were cut and stainedwith hematoxylin and eosin (H&E). The anterior and posterior chamberswere examined under light microscope and the infiltrating inflammatorycells were counted in a masked fashion. The number of infiltratinginflammatory cells in five sections per eye was averaged and recorded.EIU clinical data shown were representatives of three sets ofexperiments.

The EAU mice were euthanized and the eyes were harvested on 14th dayafter immunization, followed by fixation, paraffin embedment and stainedwith H&E. The severity of EAU was evaluated in a masked fashion on ascale of 0-4 using previously published criteria based on the number,type and size of lesions [32] and the inflammatory cells were counted asdescribed above. EAU clinical data shown were representatives of twosets of experiments, 5 animals each experimental group.

ACE2 Activity Assay

Representative retinas from each group of mice were dissected andhomogenized by sonication in ACE2 assay buffer. The ACE2 activity assaywas performed using 100 μg of retinal protein in black 96-well opaqueplates with 50 μM ACE2-specific fluorogenic peptide substrate VI (R&DSystems, Inc., Minneapolis, Minn.) in a final volume of 100 μl per wellreaction mixture. The enzymatic activity was recorded in a SpectraMax M3fluorescence microplate reader (Molecular Devices, LLC, Sunnyvale,Calif.) for 2hr with excitation at 340 nm and emission at 400 nm asdescribed previously [10]. For the sera samples, 10 μl sera were used ina 100 μl reaction. All measurements were performed in duplicate and thedata represent the mean of three assay results.

Ang-(1-7) estimation by Enzyme Immunoassay (EIA)

The level of Ang-(1-7) in plasma and retina were measured using acommercial EIA kit (Bachem, San Carlos, Calif.), according to themanufacturer's instructions. All measurements were performed induplicate and the data represent the mean of two separate assay results.

Real Time RT-PCR Analysis

Total RNA was isolated from freshly enucleated eyes using Trizol Reagent(Invitrogen, Carlsbad, Calif.) according to manufacturer's instructions.Reverse transcription was performed using Enhanced Avian HS RT-PCR kit(Sigma-Aldrich, Inc., St. Louis, Mo.) following manufacturer'sinstructions. Real time PCR was carried out on real time thermal cycler(iCycler, Bio-Rad Life Sciences, Hercules, Calif.) using iQTM Sybr GreenSupermix (Bio-Rad Life Sciences, Hercules, Calif.). The threshold cyclenumber (Ct) for real-time PCR was set by the cycler software. Optimalprimer concentration for PCR was determined separately for each primerpair. Each reaction was run in duplicate or in triplicate, and reactiontubes with target primers and those with Actin primers were alwaysincluded in the same PCR run. To test the primer efficiencies, theone-step reverse-transcriptase-PCR was run with each target primer.Relative quantification was achieved by the comparative 2^(-AACt)method 1. The relative increase/decrease of mRNA target X in theexperimental group (EG) was calculated using the control group as thecalibrator: 2^(−ΔΔCt), where ΔΔ Ct is {Ct.x [EG]−[EG]−Ct.x Actin[EG]}−{Ct.x [control]−Ct. Actin [control]}. Primer sequences used inthis study are shown in Table 1. All the reactions were repeated atleast twice.

TABLE 1 Primers used for Real-Time RT-PCR analysis Accession Gene namenumber Sequences Interleukin-6 NM_031168.1Forward: 5′-TCGGCAAACCTAGTGCGTTA-3′ (4)*Reverse: 5′-CCAAGAAACCATCTGGCTAGG-3′ (5) IL-1β NM_008361.3Forward: 5′-AAAGCCTCGTGCTGTCGGACC-3′ (6)Reverse: 5′-CAGCTGCAGGGTGGGTGTGC-3′ (7) TNF-α NM_013693.2Forward: 5′-AGGCGCCACATCTCCCTCCA-3′ (8)Reverse: 5′-CGGTGTGGGTGAGGAGCACG-3′ (9) ICAM-1 NM_010493Forward: 5′-AGATGACCTGCAGACGGAAG-3′ (10)Reverse: 5′-GGCTGAGGGTAAATGCTGTC-3′ (11) MCP-1 NM_011333Forward: 5′-CCCCACTCACCTGCTGCTACT-3′ (12)Reverse: 5′-GGCATCACAGTCCGAGTCACA-3′ (13) β-Actin X03672Forward: 5′-AGCAGATGTGGATCAGCAAG-3′ (14)Reverse: 5′-ACAGAAGCAATGCTGTCACC-3′ (15) MAS receptor NM_008552Forward: 5′-AGGGTGACTGACTGAGTTTGG-3′ (16)Reverse: 5′-GAAGGTAAGAGGACAGGAGC-3′ (17) AT1Ra NM_177322Forward: 5′-ATCGGACTAAATGGCTCACG-3′ (18)Reverse: 5′-ACGTGGGTCTCCATTGCTAA-3′ (19) AT1Rb AK087228Forward: 5′-AGTGGAGTGAGAGGGTTCAA-3′ (20)Reverse: 5′-GGGCATTGAAGACATGGTAT-3′ (21) *Numbers in parentheses are SEQID NOS.

Fundus Imaging and Assessment of EAU

Fundus assessment of EAU was performed at day 14 after EAU induction.The pupils were dilated using atropine sulfate and phenylephrinehydrochloride. The mice were anesthetized by intraperitoneal injectionof ketamine (75 mg/kg) and xylazine (5 mg/kg) mixture, and GonakHypromellose demulcent ophthalmic solution (Akorn, Inc., Buffalo Grove,Ill.) was used on ocular surface. The fundus was imaged using the MicronII small animal retinal imaging AD camera (Phoenix ResearchLaboratories, Pleasanton, Calif.). Eyes were examined for vasculitis,focal lesions, linear lesions, retinal hemorrhages and retinaldetachment. Clinical EAU scoring was performed on a scale of 0-4, asdescribed in detail previously [31]. EAU clinical data shown wasrepresentative of two sets of experiments.

Spectral Domain Optical Coherence Tomography (SD-OCT) Imaging andAssessment of EAU Mice

Mice were anesthetized and the pupil dilated as described above.Artificial tears (Systane Ultra, Alcon, Fort Worth, Tex.) were usedthroughout the procedure to maintain corneal moisture and clarity.SD-OCT images were obtained in mice on 14th day after immunization usingthe Bioptigen Spectral Domain Ophthalmic Imaging System (Bioptigen,Inc., Durham, N.C.). Images acquired by the software provided from thecompany. The average single B scan and volume scans were obtained withimages centered on optic nerve head. The retinal thickness was measuredfrom five frames of the volume of OCT images and averaged from theintensity peak of boundary corresponding to the vitreo-retinal interfaceto the intensity peak corresponding to the retinal pigmented epithelium[53]. EAU clinical data shown was representative of two sets ofexperiments.

Statistical Analysis

Data are expressed as the mean+SD of at least two independentexperiments. Differences between mean values of multiple groups wereanalyzed by one-way analysis of variance with Dunnett's test for posthoc comparisons. A p-value less than 0.05 was considered statisticallysignificant.

Results

Hyperactivity of the renin-angiotensin system (RAS) resulting inelevated Angiotensin II (Ang II) contributes to all stages ofinflammatory responses including ocular inflammation. The discovery ofangiotensin-converting enzyme 2 (ACE2) has established a protective axisof RAS involving ACE2/Ang-(1-7)/Mas that counteracts the proinflammatoryand hypertrophic effects of the deleterious ACE/AngII/AT1R axis. In thepresent example, we demonstrate that enhancing the systemic and localactivity of the protective axis of the RAS by oral delivery of ACE2 andAng-(1-7) bioencapsulated in plant cells confers protection againstocular inflammation. Both ACE2 and Ang-(1-7), fused with the non-toxiccholera toxin subunit B (CTB) were expressed in plant chloroplasts asdescribed in Example I. In the present example, we show that increasedlevels of ACE2 and Ang-(1-7) were observed in circulation and retinaafter oral administration of CTB-ACE2/Ang-(1-7) expressing plant cells.Oral feeding of mice with bioencapsulated ACE2 or Ang-(1-7)significantly reduced endotoxin-induced uveitis (EIU) in mice. Treatmentwith bioencapsulated ACE2 or Ang-(1-7) also dramatically decreasedcellular infiltration, retinal vasculitis, damage and folding inexperimental autoimmune uveoretinitis (EAU). Thus, enhancing theprotective axis of RAS by oral delivery of ACE2/Ang-(1-7)bioencapsulated in plant cells provide an innovative, highly efficientand cost-effective therapeutic strategy for ocular inflammation such asEIU and EAU.

The ability to deliver drugs efficiently to the retina or the brainremains a key challenge due to anatomic barriers and physiologicalclearance mechanisms [13]. The Plant chloroplast genetic engineeringsystem to express therapeutic proteins is emerging as a highlyefficient, cost-effective approach for therapeutic interventions of manypathologic conditions [12, 14]. In contrast to current proteinproduction systems (mammalian, yeast, or bacteria), the transplastomicsystem requires no complex production/purification steps [14]. Currentbiopharmaceuticals are not affordable to more than half of the globalpopulation because of use of prohibitively expensive production,purification and delivery systems [14]. However, chloroplasts producethe same biopharmaceuticals at a significantly lower cost by eliminatingfermentation, purification, cold chain and sterile delivery systems.Such cGMP facilities to produce plants for human clinical studies arealready in use in the US (e.g. Fraunhofer, Delaware, KentuckyBioprocessing, etc.). Ultimately, the therapeutic proteins will beprovided to patients as capsules after lyophilization of plant cells,facilitating prolonged storage at room temperature. In addition,bioencapsulation of therapeutic proteins within plant cell walls enableoral delivery by their protection in the digestive system [14,15]. Thebioencapsulated proteins that pass through the stomach are released inthe intestine with the aid of commensal bacteria [16, 17] Bacteriainhabiting the human gut have evolved to utilize complex carbohydratesin plant cell wall and are capable of utilizing almost all of plantglycans. So the gut microbes recognize, import, and digest plant cellwall consisting of cellulose, hemicellulose, and pectin. Up to 10% dailyenergy is obtained from polysaccharide fiber via the symbiotic bacterialiving in human gut. These polysaccharides are broken down to sugars andfermented to short fatty acids then absorbed by human gut. Therapeuticproteins enter circulation by receptor monosialotetrahexosylganglioside(GM1) mediated delivery when fused with the non-toxic subunit B ofcholera toxin (CTB) as the transmucosal carrier [14,18-22]. The use ofCTB as a transmucosal carrier can facilitate the transportation ofconjugated proteins into circulation through its strong binding to GM1because large mucosal area of human intestine (approximately 1.8-2.7 m2[23] facilitates CTB to bind an intestinal epithelium cell up to amaximum of 15,000 CTB [24] and the rapid turnover of cell-associated GM1receptor on the cell [25]. Furthermore, the GM1 gangliosides receptorsare also found in the plasma membranes of most cells, particularly mostabundant in the nervous system and retina [26], allowing efficientuptake of CTB fusion protein in the brain and retinal cells as observedin our recent study [12]. Considering the proven anti-inflammatoryactions of ACE2 and Ang-(1-7) and the ability of CTB to cross theepithelial barrier and facilitate neuronal uptake, we hypothesized thatoral delivery of ACE2 and Ang-(1-7) fused with CTB bioencapsulated inplant cells will enhance both systemic and local activity of theprotective axis of RAS and confer protection against ocularinflammation. In this example, we tested this hypothesis in two mousemodels of ocular inflammation. We observed that oral administration ofCTB-ACE2/Ang-(1-7) bioencapsulated in plant cells reduced ocularinflammation in both EIU and EAU models, providing proof-of-concept thatenhancing the protective axis of RAS by oral delivery of ACE2/Ang-(1-7)bioencapsulated in plant cells provides an innovative, highly efficientand cost-effective therapeutic strategy for ocular inflammation such asuveitis and autoimmune uveoretinitis.

RESULTS Creation of Transplastomic Plants Expressing CTB-ACE2/-Ang-(1-7)

The CTB-fused therapeutic genes were cloned into the chloroplasttransformation vector, pLD. The hinge site (FIG. 1) was introducedbetween CTB and therapeutic proteins to avoid steric hindrance andfacilitate formation of pentameric structure of CTB fused to therapeuticproteins, when expressed in chloroplasts. Pentameric structure of CTBplays a critical role in translocating fusion proteins into epithelialcells via the GM1 receptor. Furin cleavage site (Figure la) facilitatesrelease of therapeutic proteins from CTB after transmucosal delivery.The furin protease is ubiquitously present in all cell and tissue typesand the consensus cleavage site is well characterized [27]. Theintroduction of the consensus furin cleavage site between CTB and fusedproteins will ensure efficient release of the therapeutic proteins fromCTB into the circulation. For the site-specific integration of theCTB-ACE2/-Ang-(1-7) expression cassette into chloroplast genome, thecassette was flanked by trnl and trnA sequence, which are homologous toendogenous chloroplast sequence. Light regulated strong chloroplastpromoter,

PsbA, was used to express the fusion gene. To screen chloroplasttransformants, aminoglycoside 3′-adenylyltransferase gene (aadA) wasdriven under the control of ribosomal rRNA promoter (Prrn) to disarm theinhibitory action of spectinomycin on chloroplast translation (FIG. 1).The sequence-confirmed chloroplast transformation vectors were bombardedonto leaves to create the transplastomic plants expressing CTB-ACE2 andCTB-Ang-(1-7), using biolistic particle delivery system. Shoots emergedfrom spectinomycin containing regeneration medium were investigated forthe site specific integration of the expression cassette into thechloroplast genome, using PCR analysis. The specific primer sets weredesigned to amplify fragments in the size of ˜1.65 kb with 3P/3M forboth CTB-ACE2 and CTB-Ang-(1-7), ˜4.5 and ˜2.2 kb with 5P/2M forCTB-ACE2 and CTB-Ang-(1-7), respectively, and ˜3.0 and ˜1.1 kb with 5P/Rfor

CTB-ACE2 and CTB-Ang-(1-7), respectively. Positive shoots displaying theexpected right size fragments were subjected to two more rounds oftissue culture under antibiotic selection to achieve homoplasmy. Thehomoplasmic plants were confirmed by Southern blot analysis (data notshown), transferred and grown in a temperature- and humidity-controlledgreenhouse. The expression level of CTB-fused therapeutic proteins ofmature leaves was measured quantitatively using densitometry and Image Jor ELISA with known amount of CTB proteins to generate the standardcurve. The expression level was up to 2.14% and 8.7% of total leafprotein for CTB-ACE2 and CTB-Ang-(1-7) at their peak, respectively (datanot shown). For consistency of batches between harvests, leaves werealways harvested at 6 pm to maximize accumulation of therapeuticproteins expressed under the control of a light regulated promoter(psbA). Also, only mature leaves were chosen for harvest to maintainsimilar expression levels of the proteins between batches. While it isdifficult to precisely control expression levels at the time of harvest,dosage is precisely determined after lyophilization and the same dose isdelivered by varying the weight of lyophilized powder in each capsule orgavage.

Characterization of CTB-Fused Therapeutic Proteins Expressed inChloroplasts

CTB was used as a carrier to allow therapeutic proteins to pass throughboth epithelial and blood- retinal barrier [12], which is mediated bythe interaction between pentameric CTB and GM1 receptor. To investigatethe proper folding and assembly of the pentameric structure of CTB-fusedtherapeutic proteins in chloroplasts, western blot analysis wasperformed with CTB-Ang-(1-7). Proteins were extracted undernon-denaturing conditions, followed by either treatment with or withoutdenaturing agents, prior to separation on SDS-acrylamide gel. There wasnegligible change in polypeptide profile oligomeric structures ofCTB-Ang-(1-7) when protein was treated with DTT alone (FIG. 12a ). Incontrast, boiling samples showed dramatic change in polypeptide patterns(FIG. 12a ). This is consistent with the previous studies ondissociation of CTB pentameric structure [28]. Multiple interactionsbetween CTB monomers, such as 30 hydrogen bonds, 7 salt bridges andhydrophobic interactions, make pentameric structure of CTB highlyresistant to the dissociation so the pentamer structure is not affectedby denaturants such as SDS and DTT. However, the structure can bedissociated by using heat energy. This could be due to the difference inaccessibility of denaturing agents to their targets. The access of DTTto the intramolecular disulfide bond of monomer is not easy unlesspentameric structure dissociates first, due to the intimate interactionsbetween monomers described above. As expected, both denaturing agentsshowed no high molecular weight oligomers (pentamer-pentmerinteractions), but dimeric and monomeric forms of CTB-Ang-(1-7) wereobserved (FIG. 12a ). The intramolecular disulfide bond of CTB monomerwas easily disrupted by DTT after boiling than either boiling alone orDTT alone (FIG. 12a ). Boiling allowed easy access of DTT to theinternal disulfide bond by breaking intimate interactions between CTBmonomers (hydrogen bonds and salt bridges) (FIG. 12a ). From theseresults, it is evident that the disulfide bond of CTB-Ang-(1-7) monomerwas properly formed and the interactions between the monomers ofpentameric structure of CTB-Ang-(1-7) were well established inchloroplasts.

For clinical application, long-shelf life and stability of therapeuticprotein expressed in plants are very important for successful andcost-effective treatment. Therefore, lyophilized CTB-fused therapeuticprotein leaves were fully characterized. The weight of lyophilized leafis usually reduced by 90% to 95% as a result of removal of water fromplant cells, leading to more total protein per mg of leaf powder.Extraction of concentrated proteins from lyophilized leaf materialsneeds more volume of the extraction buffer because the amount of waterlost in the process of lyophilization is slowly reabsorbed by the driedmaterials. For quantitation of lyophilized leaf materials, 10 mg oflyophilized powered leaf materials were resuspended in 300 μl extractionbuffer in contrast to 100 mg of fresh leaf materials in the same volumeof extraction buffer. Then the extracted total proteins were used forquantification for comparison between fresh and lyophilized leafmaterials. Western blot analysis of the CTB-Ang-(1-7) showed that theband patterns between fresh and 3-month old lyophilized leaf materialswere identical (FIG. 12b ), confirming stability during lyophilizationand prolonged storage at room temperature. As seen in the blot,twenty-time less lyophilized protein sample loaded showed similar bandintensities to fresh leaf proteins (FIG. 12b ). The quantity ofCTB-Ang1-7 in fresh and lyophilized leaves was measured usingimmunoblots (FIG. 12b ) and Image J software; the quantity ofCTB-Ang-(1-7) increased 14.3 times in lyophilized leaves when comparedto fresh leaves (FIG. 12c ). The lyophilized CTB-ACE2 leaves showed 20.5fold more CTB-Ace2 than fresh leaves when quantified using ELISA (FIG.12d ).

In this study, we observed that there was no damage or loss of thefusion protein (FIG. 12b ) under the optimized lyophilizationconditions. Moreover, the intactness of the pentameric structure of thelyophilized CTB-fused proteins was well preserved up to 15 months atroom temperature, as confirmed in GM1 binding assay which showed bindingaffinity of the lyophilized CTB-fused proteins to GM1 as compared torespective positive control, CTB (FIG. 12e ). Taken together, thehomoplasmic transplastomic plants expressing CTB-ACE2 and -Ang-(1-7)were created and the fusion protein was properly expressed, folded, andassembled in chloroplasts. The folding, assembly and functionality oftherapeutic proteins were well preserved in lyophilized leaves.

ACE2 activity assay using protein extracts isolated from plant leavesshowed that plant cell expressed human ACE2 is enzymatically active(FIG. 13a ). To investigate the in vivo potential of CTB-ACE2 andCTB-Ang-(1-7) to cross the intestinal barrier and tissue uptake, wildtype C57B1/6J mice were fed with either fresh (F, ˜500 mg/mouse), orten-fold less lyophilized (L, ˜50 mg/mouse) CTB-ACE2, or controluntransformed (WT) leaf materials for three days, mice were sacrificedat 5hr after the last gavage. Circulatory and retinal ACE2 and Ang-(1-7)levels were measured by ACE2 activity assay, EIA and Western blotting(FIG. 13). ACE2 protein can be detected in both serum and retina 5 hoursafter oral gavage (FIG. 13b ). Oral administration of either freshfrozen (F) or lyophilized (L) CTB-ACE2 transgenic leaf materialsresulted in an increase of approximately 40% and >20% in ACE2 enzymaticactivity in serum and retina, respectively when compared to WT leaf fedmice (FIG. 13c ). There was a 15% increase in plasma and nearly 50%increase in Ang-(1-7) peptide level in the retina in CTB-Ang-(1-7)expressed leaf material fed group, detected by Ang-(1-7) specific EIAkit (FIG. 13d ).

Oral Administration of Bioencapsulated CTB-ACE2 and CTB-Ang-(1-7)Reduced the Infiltration of Inflammatory Cells Induced by EIU

We next examined the effects of ACE2 and Ang-(1-7) on endotoxin-inducedinfiltration of inflammatory cells such as leucocytes and monocytes inthe iris, ciliary body, anterior chamber, and posterior chamber of theeye. Sagittal sections were stained with H&E and examined under brightfield microscope. The histological evaluation of LPS injected eyes frommice fed with WT leaf revealed severe signs of uveitis with massiveinfiltration of inflammatory cells into the iris and ciliary body (ICB)(140±21 cells/section), anterior chamber (265±52 cells/section) and theposterior chamber (202±37 cells/section) (FIG. 14). Prophylactictreatment with CTB-ACE2 showed significantly diminished uveitis andreduced number of inflammatory cells into the ICB (60±09 cells/section),anterior chamber (96±15 cells/section) and also into the posteriorchamber (82±15 cells/section). Similar results were observed in ICB(70±15 cells/section), anterior chamber (114±36 cells/section) and theposterior chamber (28±15 cells/section) when animals pretreated withCTB-Ang-(1-7) expressed leaf material (FIG. 14).

The therapeutic effect of different doses of CTB-ACE2 in EIU wasevaluated using lyophilized leaf materials. Oral feeding of lyophilizedCTB-ACE2 at 50 mg/day significantly prevented inflammatory cellinfiltration into the iris and ciliary body, anterior chamber andposterior chamber to the same extent as the fresh leaf material at 500mg/day; CTB-ACE2 feeding at 25mg/day had moderate but significantprotection, whereas 12.5 mg/day did not show any protective effect inEIU (FIG. 14C).

Oral Administration of Bioencapsulated CTB-ACE2 and CTB-Ang-(1-7)Reduced the

Expression of the Inflammatory Cytokines in EIU Eyes To investigate theeffects of ACE2 and Ang-(1-7) on the expression of inflammatorycytokines in EIU eyes, the mRNA levels of cytokine genes were determinedby real-time RT-PCR. In the WT leaf fed mice, LPS caused a significantincrease in mRNA levels of Interleukin-6 (IL-6), Interleukin-1β(IL-1(3), Tumor necrosis factor-α (TNF-α) and vascular endothelialgrowth factor (VEGF) and this increase was reduced in mice fed with ACE2or Ang-(1-7) leaf materials (FIG. 15a ). These results suggest that ACE2and Ang-(1-7) reduced infiltration of inflammatory cells and cytokineproduction through suppressing their gene expressions during EIU. Toinvestigate the molecular mechanisms of leucocyte recruitment, the mRNAlevels of intercellular adhesion molecule-1 (ICAM-1) and monocytechemoattractant protein (MCP-1) were measured in EIU eyes. Theexpression of both ICAM-1 and MCP-1 was significantly increased in LPSinduced EIU eyes in mice treated with WT leaf material and wassignificantly reduced in mice fed with CTB-ACE2 or CTB-Ang-(1-7) (FIG.15a ).

The Impact of Oral Administration of Bioencapsulated CTB-ACE2 andCTB-Ang-(1-7) on the Expression of the Retinal RAS Genes During EIU

In addition to circulating RAS, all components of RAS have been detectedin the retina and a local retina RAS may play an important role inmodulating local immune responses [10,11,29]. We compared ocular mRNAlevels of the key RAS genes in animals fed with ACE2 and Ang-(1-7)expressing leaf materials as well as untransformed WT leaf materials.LPS-induced EIU resulted in increased expression of both ACE and ACE2,however prominent increase in ACE (more than 4-fold increase) than ACE2(less than 2-fold increase), resulted in increased ratio of ACE/ACE2(FIG. 15b ). CTB-ACE2 or CTB-Ang-(1-7) oral feeding normalized the shiftof ACE/ACE2 ratio (FIG. 15b ). The mRNA levels for receptors for Ang II(AT1Ra, AT1Rb) were also increased in EIU mice fed with control leafmaterial (˜4-fold and 1.7-fold respectively) (FIG. 15b ), both of whichwere significantly decreased in mice fed with CTB-ACE2 or CTB-Ang (1-7).There was a slight but significant increase in Mas, the receptor forAng-(1-7). Interestingly the Mas mRNA level in the retina was furtherincreased in mice fed with CTB-ACE2 (-2-fold increase), and even moreincrease in mice fed with CTB-Ang-(1-7) leaf material (-3-fold increase)(FIG. 15b ), suggesting a possible feed-forward regulatory response inlocal retinal RAS.

Oral Delivery of Bioencapsulated CTB-ACE2 and CTB-Ang-(1-7) AttenuatedAutoantigen Induced Uveoretinitis

Experimental autoimmune uveoretinitis was induced in an autoimmunesusceptible B10.RIII mouse strain by active immunization using a peptidederived from the retinal protein IRBP [30]. Evident inflammatoryreactions such as mild to severe vasculitis, focal lesions, largeconfluent lesions, retinal hemorrhages and folding, corneal edema etc.,were observed in WT leaf fed animals by fundoscopy examination at day 14of EAU induction (FIG. 16A, a-b). However, mice fed with CTB-ACE2 or-Ang-(1-7) leaf materials showed significantly reduced inflammatoryreactions (FIG. 16A, c-d, and e-f). The clinical scoring, using thecriteria reported by Copland et al [31], showed that eyes from CTB-ACE2or CTB-Ang-(1-7) fed animals had significantly improved clinical scores(EAU grade, 2.3±1.2 and 2.3±1.3 respectively) compared to eyes fromanimals fed with WT leaf (EAU grade, 3.4±0.53) (FIG. 16B).

The uveoretinitis was also evaluated by OCT imaging on day 14 afterimmunization with IRBP. In few cases severe retinal pathology such ashigh level of cellular infiltration, edema, folds, and hemorrhageslimited OCT resolution of retinal layers. In most cases, intravitrealcellular infiltration, retinal vasculitis, disorganized retinal layersand increased retinal thickness due to retinal folds and edema, can beeasily visualized with OCT imaging as shown in FIG. 17a in untreated orWT leaf material treated animals, these pathologies are much improved inmice treated with CTB-ACE2 or Ang-(1-7) (FIG. 17a ). Treatment withCTB-ACE2 or CTB-Ang-(1-7) leaf materials significantly reducedEAU-induced increased retinal thickness (269±32 μm and 241±52 μmrespectively) compared to eyes treated with WT leaf (316±32 μm) (FIG.17b ).

Oral Administration of CTB-ACE2 and CTB-Ang-(1-7) AmelioratesHistological Findings in the EAU Mice

Histological examination on day 14 showed a severe intraocularinflammation evidenced by massive infiltration of inflammatory cells,intensive retinal vasculitits, and changes in the retinal thickness,folding of retina, as well as photoreceptor damage in the WT leaf fedmice (FIG. 18a ). However, only scattered inflammation of inflammatorycells and minor retinal folding was observed in CTB-ACE2 orCTB-Ang-(1-7) treated animals (FIG. 18a ). Histopathological grading,using the criteria reported by Thurau et al.[32] showed that WT leaf fedeyes (EAU grade, 2.95±0.717) had significantly severe inflammation ascompared to CTB-ACE2 (EAU grade, 1.1±0.616) and CTB-Ang-(1-7) (EAUgrade, 0.92±0.535) expressed leaf fed eyes (FIG. 18b ). Similarlysignificantly higher numbers of inflammatory cells were observed in theposterior chamber of WT leaf fed mice compared to the CTB-ACE2/Ang-(1-7)expressed leaf fed mice (FIG. 18b ).

To determine whether CTB-Ang-(1-7) treatment can also improve EAU afterits onset or during its progression, daily oral feeding was delayed today 5 and day 10 after EAU induction, and continued to day 14 when micewere euthanized for evaluation. We observed that feeding from day 5onward up to day 14 after IRBP injection is equally as effective asfeeding from day 0, but feeding started at 10 after EAU induction had noimprovement of ocular pathology (FIG. 19).

DISCUSSION

In this study, we have developed an expression system to generate highlevels of human ACE2 and Ang-(1-7) within plant chloroplasts usingtransplastomic technology. Oral gavage of plant cells expressing ACE2and Ang-(1-7) fused with CTB in mice resulted in increased circulatingand retinal levels of ACE2 and Ang-(1-7), reduced ocular inflammation intwo different models: endotoxin-induced uveitis (EIU) and autoantigeninduced experimental autoimmune uveoretinitis (EAU).

Among many advantages of transplastomic technology, the high copy numberof a transgene, up to >10,000 copies per cell, is a key to successfulhigh level expression of therapeutic proteins in chloroplasts. However,this advantage could be limited when human transgenes are notcodon-optimized because the preference of codon usage of chloroplast isdifferent from that of eukaryotic cell. The codon adjustment forchloroplast expression system is crucial for the efficient expression ofhuman genes [33]. So, the relatively low expression ACE2 over theAng-(1-7) is probably due to the use of native human gene sequence (805amino acids). However, several other tools were incorporated into oursystem to offset low expression of the transgenes so that the contentsof therapeutic proteins expressed in chloroplasts can be increased. Forexample, the expression of therapeutic proteins under the control oflight-regulated strong chloroplast promoter, harvest of mature leaves atthe end of day, and lyophilization of the harvested leaves. Thechloroplast psbA promoter is light regulated and therefore harvestingleaves before sunset maximizes accumulation therapeutic proteins.

Moreover, the amount of therapeutic proteins in plant leaves can beconcentrated by lyophilization (FIGS. 12c and 12d ). The process ofdehydration of the leaves under vacuum at −51° C. for 3 days cansignificantly reduce the risk of microbial contamination [14]. Thelong-term shelf life of the lyophilized proteins at room temperature canalso decrease the cost associated with the cold chain of currentinjectable proteins [14]. The effect of lyophilization on the stabilityof proteins expressed in chloroplasts has been extensively studied inour lab under the condition at which temperature and pressure were setup at −51° C. and 27 mTorr, respectively, according to the chart ofvapor pressure of ice provided by manufacturer. When the effect ofduration of lyophilization was investigated, large protective antigen(PA, 83kDa) expressed in lettuce chloroplasts was stable at 24, 48, and72 hrs of lyophilization. In addition, the lyophilized antigen was morestable at room temperature than the commercially purified antigensstored at low temperatures. Further, the lyophilized PA was found to bestable up to 15 months at room temperature without any degradation [14].Other transplastomic plants expressing CTB-exendin 4 [21] and CTB-FactorVIII [34] showed similar stability of fusion proteins and protection oftheir assembly, folding and disulfide bonds similar to fresh leaves.

Drug delivery to different compartments of the eye, particularly to theposterior segment of the eye, is a major challenge due to severalbarriers formed by both anatomical structure and the protectivephysiological mechanisms of the eye[13]. Large molecular weighttherapeutics such as peptides/proteins and oligonucleotides aredelivered mostly via intravitreal route. However, frequentadministration via this route is often associated with manycomplications such as retinal detachment, endophthalmitis, and increasedintraocular pressure [35, 36]. We demonstrate that oral administrationof CTB-ACE2 increased ACE2 activity in sera and retina. Similarlyincreased level of plasma and retinal Ang-(1-7) was observed when theanimals were orally administered with CTB-Ang-(1-7), as observed inprevious study of oral delivery of bioencapsulated proteins acrossblood-brain and retinal barriers [12]. The increased level of theAng-(1-7) could be stemmed from the fusion with CTB. The short peptideof Ang-(1-7) fused to CTB becomes stabilized in a form of pentamericstructure in plant cells (FIG. 12a ). However, only monomers areobserved in sera once delivered into circulation. Considering that theefficiency of furin cleavage site depends on the flanking amino acidsequence of the fused protein [37], Ang-(1-7) fusion to CTB did notoffer optimal furin cleavage site. Thus, the cleavage between CTB andAng-(1-7) is not likely to be fast or efficient once the fusion proteingets into sera. In addition, the CTB fusion provides N-terminalprotection for Ang-(1-7) so its stability is extended for several hours,while injectable Ang-(1-7) has a very short half-life in sera [38, 39].Although the Ang-(1-7) level was increased in both plasma and retina(FIG. 13d ), the level of Ang-(1-7) increase in plasma was less than inretina (FIG. 13d ). This difference could be attributed to the increasedretention in tissues (cells) and to their rapid clearance in the sera byproteases. Similar result was also observed in our previously publishedstudy in which GFP level was shown several fold higher in tissues thanin sera [12,19]. Although EIU was originally used as a model of anterioruveitis because of its characteristic infiltration of leucocytes intothe anterior chamber of the eye [4], growing evidences suggest that italso involves inflammation in the posterior segment of the eye, withrecruitment of leucocytes that adhere to the retinal vasculature andinfiltrate the vitreous cavity [3]. Our results demonstrate thatenhanced level of ACE2/Ang-(1-7) in both circulation and ocular tissuessuppressed the endotoxin-induced ocular inflammation, which is evidentfrom significantly reduced number of infiltrating inflammatory cells inthe iris-ciliary body, anterior and posterior chambers. This result isconsistent with studies showing anti-inflammatory property ofACE2/Ang-(1-7) in other disease models [9]. The dose dependent studyusing bio-encapsulated lyophilized CTB-ACE2 in EIU model furtherconfirmed that a dose of ˜50 mg/day for four days can significantlyprevent the endotoxin- induced inflammation. We also showed thatincreased ACE2/Ang-(1-7) significantly suppressed the LPS-induced ocularexpression of IL-6, IL-1β TNF-α and VEGF. It has been reported that inEIU model, leucocytes are markedly attracted to inflamed ocular tissuessuch as the iris [40], vitreous cavity [41] and retina [42], withneutrophils and macrophages being major leucocyte constituents. MCP-1 isknown as one of the important factors for leucocyte recruitment, and isup-regulated during EIU [43] whereas ICAM-1 is known to be the keymolecule of leucocyte adhesion and/or transmigration [44]. Our studydemonstrates that mice fed with ACE2/Ang-(1-7) leaf materials showeddecreased ocular expression of MCP-1 and ICAM-1 in EIU eyes,contributing to the diminished inflammatory response by inhibitingleucocyte recruitment and adhesion in the ocular tissue. These resultsare consistent with the histopathology observation that LPS-inducedacute inflammation caused the increase of inflammatory cell recruitment,while ACE2/Ang-(1-7) treatment significantly reduced the inflammatorycells in iris/ciliary body, anterior and posterior chamber.

It has been shown that ACE2/Ang-(1-7) may directly reduce inflammatoryresponses in immune cells such as macrophages [45]. Our study also showsmodulatory ability of ACE2/Ang-(1-7) on local immune response andcytokine/chemokine expression. In fact the Mas receptor is expressed notonly in retinal vascular cells, astrocytes and muller glia, but also inretinal neurons, consistent with its role in neuro-vascular and immuneresponse modulation. Moreover, our results show that eyes with EIU areassociated with decreased expression of ACE2 and Mas receptor andincreased expression of vasoconstrictive axis genes such as ACE, AT1Ra,AT1Rb during EIU. This is prevented by ACE2/Ang(1-7), suggesting thatthe anti-inflammatory effect of ACE2/Ang-(1-7) may be associated withMas receptor and ACE2 up-regulation, and down-regulation of ACE andAT1Ra/AT1Rb, resulting in reduction of ocular inflammation.

In this study, we also investigated the effect of oral administration ofCTB-ACE2/Ang-(1-7) on the development of EAU in mice and showedsignificant improvement of EAU eyes in mice treated withCTB-ACE2/Ang-(1-7). The pathogenesis of EAU is different from EIU. EAUis defined primarily as posterior segment disease as the target antigensreside in the retina and characterized by cellular infiltrates, retinalfolds, detachment, granulomatous infiltrates in the retina and choroid,vasculitis, retinal neovascularization, mild to severe photoreceptorloss [32]. Histopathological examination confirmed a significant overallreduction of disease severity in the CTB-ACE2/Ang-(1-7) treatment groupsevaluated by non-invasive funduscopy and OCT imaging methods.Furthermore, the retinal detachment, photoreceptor layer damage,infiltration of inflammatory cells was markedly prevented byCTB-ACE2/Ang-(1-7) treatments. Some of the fundoscopicallynormal-looking eyes showed few foci of very mild cellular infiltrates onhistological evaluation. This is consistent with the findings from OCTimaging, demonstrating a better correlation of histological findings andpathological changes revealed by non-invasive OCT imaging in the retinaduring EAU. Thus, oral administration of CTB-ACE2/Ang-(1-7) from theinduction to peak of EAU was able to ameliorate the progression ofdisease evaluated by clinical funduscopic score, OCT imaging andhistopathological observation. Moreover, delayed oral administration ofCTB-ACE2/Ang-(1-7) from day 5 after EAU induction was also able todecrease the progression of EAU.

Increasing evidence has shown that shifting the balance of RAS towardsthe protective axis by activation of ACE2 or its product, Ang-(1-7) isbeneficial and anti-inflammatory [9,46]. Our findings also demonstratethat oral administration of CTB-ACE2/Ang-(1-7) provides robustprotective anti-inflammatory effects against the pathophysiology in bothEIU and EAU models.

In conclusion, this study provides proof-of concept for production oftherapeutically active ACE2/Ang-(1-7) bioencapsulated in plant cells forcost effective oral therapy for ocular applications and enhancingACE2/Ang-(1-7) using this approach may provide a new avenue and a noveltherapeutic strategy for the treatment of acute uveitis, autoimmuneuveoretinitis and other ocular diseases.

REFERENCES FOR EXAMPLE 2

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EXAMPLE 3 Codon Optimization of ACE2 and Ang-(1-7)

In a recent study, Nakamura et al. disclose the importance ofcompatibility between the psbA 5′-UTR and its 5′ coding sequence whenusing codon-optimized heterologous genes. Previously reported codonoptimization studies used only smaller eukaryotic coding sequences (<30kDa). However, there is a great need to express larger human genes (eg.Human blood clotting factor VIII >200kDa) that would requireoptimization of not only codons but also compatibility with regulatorysequences for optimal translation initiation, elongation and greaterunderstanding of tRNAs encoded by the chloroplast genome or importedfrom the cytosol. However, no systematic study has been done to utilizethe extensive knowledge gathered by sequencing several hundredchloroplast genomes to understand codon usage and frequency of highlyexpressed chloroplast genes.

Among the 140 transgenes expressed in chloroplasts, >75% use the psbAregulatory sequences (Daniell et al., 2016). Most importantly,compatibility between the 5′-UTR of psbA and its coding region isimportant for efficient translation initiation (Nakamura et al., 2016).For these reasons, a new codon optimization program was developed usingcodon usage of the psbA genes from 133 sequenced chloroplast genomes(FIGS. 20A and 20B). We first investigated expression of synthetic genesusing only the most highly preferred codon for each amino acid, which isreferred to as the “old” version in this study. When this resulted ineven lower levels of expression than the native gene (see data presentedbelow), a “new” codon optimizer algorithm was developed using codonusage hierarchy observed among sequenced psbA genes. See FIGS. 20C, 20D,and 20E. Therefore, most of the rare codons in heterologous genes weremodified based on codons with >5% frequency of use in the psbA genes.

In this study, the CNTB coding sequence was not codon-optimized becauseof its prokaryotic origin and high AT content (65.37%). Mostimportantly, the expression level of CNTB (native sequence) fused withproinsulin in tobacco chloroplasts reached up to 72% of total leafprotein (Ruhlman et al., 2010) and 53% of total leaf protein in lettucechloroplasts (Boyhan and Daniell, 2011), indicating that there is nolimitation on translation of the CNTB coding sequence in chloroplasts.

When the psbA-based codon table is compared with total chloroplast codonusage tables, which are generated based on all chloroplast genes ofLactuca sativa (57,528 codons from 189 coding sequences) or Nicotianatabacum (34,756 codons from 137 coding sequences) (Nakamura et al.,2000), there was no significant difference in AT content of codingsequences; it varied between 59.59% and 61.76%. However, there arestriking differences between psbA-based and total chloroplast gene-basedcodon tables when individual codons are compared. Ten rare codonsidentified in the psbA codon table (<5% usage frequency), (which werenot used in codon-optimized sequences in the new version) were notidentified as rare codons in the total chloroplast gene codon usagetable. For example, CTC (leucine) is almost never used in psbA genes(0.1%) but this codon's usage is >6.4% in the lettuce or 7.5% in thetobacco total chloroplast codon table. Likewise, CGG (arginine) and ACG(threonine) are used with frequencies of 0.5% and 0.8%, respectively, inthe psbA-based codon table, but the same codons are used with 7.6% and9.6% frequency in lettuce or 8.3% and 10.8% frequency in tobacco totalcodon tables. Inadequate identification of rare codons in totalchloroplast gene codon tables could be due to the averaging effect ofcombining highly and poorly expressed genes regardless of theirtranslation efficiency in chloroplasts. This is true for the hierarchyin codon usage among synonymous codons. For example, the 5th codon inthe hierarchy for leucine is CTG, which is used at a frequency of 3.7%in the psbA-based codon table; the difference in usage between the 6thcodon (CTC, 0.1%) is 37 fold. However, there is no difference in thepercentage of codon usage between 5th and 6th codons in lettuce ortobacco chloroplast gene codon tables; the lettuce codon table shows6.7% (CTG, 5th) and 6.4% (CTC, 6th) frequency and the tobacco tableshows 7.5% (5th codon) and 7.1% (6th codon) frequency. The disadvantageof using a total chloroplast gene-based codon table is quite obvious:real differences in codon preference in translation are masked, asreported previously (Surzycki et al., 2009).

Expression of Angiotensin Converting Enzyme in Lettuce Chloroplasts

Because of low expression level of native ACE2 in transplastomic plants,the creation of a codon optimized ACE2 polypeptide is described in thepresent example. To improve the production ACE2 in chloroplasts, wefirst developed new software for codon optimization by creating adatabase for codon usage of psbA genes from 133 plant species and analgorithm was developed to replace rare codons with preferred codons.The chloroplastpsbA gene was chosen as a model because this is the mosthighly expressed chloroplast gene. The psbA based codon table showed nosignificant difference in AT content of coding sequences when comparedto total chloroplast codon usage tables generated using all lettuce(57,528 codons from 189 coding sequences). However, there are majordifferences between a single gene based codon table and total gene basedcodon tables in the usage frequency of individual codons and hierarchyin codon usage. Further, eleven codons identified as rare codons in psbAgene based codon table were not identified as rare codons in chloroplasttotal gene based codon tables. For example, CTC for leucine is almostnot used in psbA genes (0.1%) but the same codon is used at thefrequency of 6.4% in lettuce; CGG for arginine in psbA genes (0.5%) butat the frequency of 7.6% in lettuce total chloroplast. Thus,codon-optimized ACE2 were optimized by changing the rare codons andcodons usage frequency to resemble the chloroplastpsbA gene (FIGS. 20Aand 20B). 481 codons including 59 rare codons out of 805 codons of ACE2were replaced according to the psbA codon distribution (FIGS. 20C, 20D,and 20E). As a result, AT content of the ACE2 gene was increased from57% to 62.46%, and the frequency of codon usage is similar to psbA gene(FIG. 21A, native, FIG. 21B, optimized). Optimized sequences were thensynthesized by GenScript Inc. Genetically modified lettuce lines werecreated by bombardment of chloroplast vectors into sterile leaves usingthe gene gun. Transplastomic lettuce shoots confirmed by PCR were cutand subjected to two further rounds of selection. After selection,transplastomic lettuce plants were transferred to greenhouse (FIG.22A-22D). Total leaf protein was extracted from native andcodon-optimized transplastomic plants and the protein expression levelwas compared by Western blot analysis. The transplastomic lettuce plantsexpressing codon-optimized ACE2 showed 7.7-fold higher expression thanthe native gene (FIG. 23).

Conclusion

The present example provides a codon optimized CTB-ACE2 polypeptidesequence which is expressed at significantly higher levels in lettucechloroplasts. This increased expression should enhance the beneficialcardioprotective effects described in Example 1 and improve the antiinflammatory effects described in Example 2.

While a number of embodiments have been shown and described herein inthe present context, such embodiments are provided by way of exampleonly, and not of limitation. Numerous variations, changes andsubstitutions will occur to those of skilled in the art withoutmaterially departing from the invention herein. For example, the presentinvention need not be limited to best mode disclosed herein, since otherapplications can equally benefit from the teachings. Also, in theclaims, means-plus-function and step-plus-function clauses are intendedto cover the structures and acts, respectively, described herein asperforming the recited function and not only structural equivalents oract equivalents, but also equivalent structures or equivalent acts,respectively. Accordingly, all such modifications are intended to beincluded within the scope of this invention as defined in the followingclaims, in accordance with relevant law as to their interpretation.

1-28. (canceled)
 29. A method for the reducing proinflammatory cytokinesin the lung and reducing proliferation of vascular smooth muscle cellsin a subject in need thereof comprising oral administration of atherapeutically effective amount of; i) a fusion protein comprisingangiotensin-converting enzyme 2 (ACE-2), and cholera non-toxic B subunit(CTB); or ii) a fusion protein comprising angiotensin-(1-7)(Ang-(1-7)),and cholera non-toxic B subunit (CTB); or i) and ii) in combination in abiologically acceptable carrier, said administration being effective toreduce lung proinflammatory cytokines and proliferation of vascularsmooth muscle cells in said subject, said method optionally comprisingassessing said reduction of said proinflammatory cytokines in saidsubject.
 30. The method of claim 29, wherein the fusion protein of i)and, or ii) is produced in chloroplasts of a transplastomic plant. 31.The method of claim 30, wherein the plant is homoplasmic and leavesobtained from said homoplasmic plant are lyophilized and formulated intoa therapeutic composition comprising said angiotensin-converting enzyme2 (ACE-2), and cholera nontoxic B subunit (CTB) fusion protein.
 32. Themethod of claim 31, wherein the ACE-2 in said leaves is encoded by SEQID NO:
 24. 33. The method of claim 32, wherein the leaves furthercomprise the angiotensin-(1-7) (ANG-(1-7)), and cholera nontoxic Bsubunit (CTB) fusion protein.
 34. The method of claim 32, where saidtherapeutic composition further comprises synthetic angiotensin-(1-7).35. The method of claim 29, wherein said plant is selected from thegroup consisting of lettuce, carrots, cauliflower, cabbage, low-nicotinetobacco, spinach, kale, and cilantro.
 36. A method for the reducing ATR2levels in a subject in need thereof comprising oral administration of atherapeutically effective amount of; i) a fusion protein comprisingangiotensin-converting enzyme 2 (ACE-2), and cholera non-toxic B subunit(CTB); or ii) a fusion protein comprising angiotensin-(1-7)(Ang-(1-7)),and cholera non-toxic B subunit (CTB); or i) and ii) in combination in abiologically acceptable carrier, said administration being effective toreduce ATR2 levels in said subject, said method optionally comprisingassessing said reduction of said ATR2 levels in said subject.
 37. Themethod of claim 36, wherein the fusion protein of i) and, or ii) isproduced in chloroplasts of a transplastomic plant.
 38. The method ofclaim 37, wherein the plant is homoplasmic and leaves obtained from saidhomoplasmic plant are lyophilized and formulated into a therapeuticcomposition comprising said angiotensin-converting enzyme 2 (ACE-2), andcholera nontoxic B subunit (CTB) fusion protein.
 39. The method of claim38, wherein the ACE-2 in said leaves is encoded by SEQ ID NO:
 24. 40.The method of claim 39, wherein the leaves further comprise theangiotensin-(1-7) (ANG-(1-7)), and cholera nontoxic B subunit (CTB)fusion protein.
 41. The method of claim 39, where said therapeuticcomposition further comprises synthetic angiotensin-(1-7).
 42. Themethod of claim 36, wherein said plant is selected from the groupconsisting of lettuce, carrots, cauliflower, cabbage, low-nicotinetobacco, spinach, kale, and cilantro.